Document 1754940

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Downstream Processes: Advances in Chromatographic Separations Fundamentals and Processing
Downstream Processes
Advances in Chromatographic Separations:
Fundamentals and Processing
8:30 a.m.
Room# 16A
K. Eriksson, C. Gillespie, A. Lenhoff Papers 1-8
K. Eriksson, C. Gillespie, A. Lenhoff Papers 1-8
BIOT 1 – 8:30 a.m.
8:30 a.m Room# 16B
Upstream Processes: Advances in Biocatalysis
K. Jones Prather, M. Truppo Papers 9-16
8:30 a.m. Room# 17A
Advances in Biofuels Production: Biomass Pretreatment and Hydrolysis
Cosponsored by CELL
Financially supported by European Polysaccharide Network of Excellence
J. Zhu, Y. Kim, M. Ladisch Papers 17-24
8:30 a.m.
Room# 25A Stem Cells and Tissue Engineering:
Embryonic and Induced Pluripotent Stem Cells
R. Rao, W. Grayson Papers 25-31
8:00 a.m. Room# 25B
Biophysical & Biomolecular Processes:
Protein Conjugates: From Basic Principles to Clinically Active Drugs
Session Sponsored by LIFE
C. Meares, P. Senter Papers 32-39
11:30 a.m. Room# 16A
DIC Wang Biochemical in Engineering Award Lecture
Mobile phase modifier effects in multimodal chromatography:
Fundamental understanding and applications in bioprocessing
Melissa A Holstein1, [email protected], Siddharth Parimal1, Scott A
McCallum2, Steven M Cramer1. (1) Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180,
United States (2) Department of Biology, Rensselaer Polytechnic Institute,
Troy, New York 12180, United States
While the unique selectivities, broad applicability, and enhanced
separation power of multimodal chromatographic resins have resulted in more widespread use of these materials in recent years,
the separation of proteins with similar properties and retention behavior continues to be a challenging problem. A multifaceted approach consisting of chromatography, nuclear magnetic resonance,
and molecular dynamics simulations is used to provide fundamental insight into protein adsorption in multimodal chromatographic
systems. A wide experimental space is examined using column
and high-throughput batch chromatography experiments with a
variety of mobile phase modifiers. These experimental techniques
are used in conjunction with molecular dynamic simulations to
understand protein-ligand interactions at the molecular level by
investigating the synergy between electrostatic and hydrophobic
interactions present in a multimodal system. The results demonstrate how the appropriate use of mobile phase modifiers and multimodal resins can enable unique separations to be performed that
cannot be achieved with traditional methods.
BIOT 2 – 8:50 a.m.
Enhancement of monoclonal antibody purification processes
using multimodal chromatography
to grow, optimizing mAb production processes has become more
important in improving efficiencies and reducing cost-of-goods in
large scale manufacturing. At the same time, recent advances in
the design of multimodal chromatography resins have produced
adsorbents which provide alternative and improved selectivity as
compared to single mode chromatography resins. Utilization of
multimodal chromatography in mAb purification processes to enhance removal of product-related and process-related impurities is
quickly being adopted by the biopharmaceutical industry.
In this study, Capto adhere, a multimodal chromatography adsorbent with functionalities of ionic interaction, hydrogen bonding
and hydrophobic interaction, was evaluated for its selectivity using
multiple mAbs with different physiological properties in their purification processes. In addition, well plate-based high throughput
screening methodology was used to gain further understanding of
the interaction between antibody molecules and multimodal resins under different operating conditions. Results will be presented
demonstrating how versatile selectivity can be achieved by operating the multimodal chromatography step at different operating
conditions.
BIOT 3 – 9:10 a.m.
Investigating the use of column inserts to achieve better
chromatographic bed support
Tian Lan, [email protected], Spyridon Gerontas, Martina Micheletti,
Nigel Titchener-Hooker.Department of Biochemical Engineering, University
College London, London, United Kingdom
Chromatography plays an important role in the downstream processing of proteins. Over the past years there has been a steady
move toward the adoption of more rigid, porous particles in order
to combine ease of manufacture with increased levels of productivity. The latter is still constrained by the onset of compression
where the level of wall support becomes incapable of withstanding
flow-induced particle drag. In this study we investigate how, by the
installation of cylindrical column inserts, it is possible experimentally to enhance the level of wall support. Experiments were conducted to examine the effect of the position of the insert in the column and its dimensions on the critical velocity at which the onset
of compression occurs. It was found that when an insert installed
at the bottom (downward flow), it can provide about 30% increase
in critical velocity without significant affecting column efficiency.
Junfen Ma, [email protected], Xiaoyang Zhao, Hai Hoang, Gina
Sperrazzo, Rafael Gonzalez, Tony Hong, Bryan Dransart, Tim Tressel.
Purification Process Development, Amgen Inc., Thousand Oaks, CA 91320,
United States
As monoclonal antibody (mAb) therapeutics market continues
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Sunday Morning
Sunday Morning
Sessions
Sunday Morning Sessions
BIOT 6 – 10:30 a.m.
BIOT 8 – 11:10 a.m.
Rapid development of cation exchange chromatography step
elution for monoclonal antibody purification
Understanding chromatography fouling in vaccine and
therapeutic protein manufacture
Edward Close1,2, Jing Jin1, Jeffrey Salm3, Eva Sørensen2, Daniel G
Bracewell1, [email protected]. (1) Department of Biochemical
Robustness of on-column trisulfide elimination from protein
therapeutics
Lucian Young, [email protected], Katherine Cai, Amit Varma,
Ping Y. Huang.Department of Process Sciences, Abbott Biotherapeutics
Corporation, Redwood City, CA 94063, United States
Cation exchange (CEX) chromatography is widely used in antibody platform purification processes for aggregate and other impurity removal. Step elution for CEX is preferred at manufacturing scale. However, each antibody is unique and the identification
of the salt concentration for step elution is often time consuming.
We have developed a rapid linear gradient method to determine
the salt concentration for step elution and it was successfully demonstrated in several different antibodies. Design of experiment
(DOE) further proved that the determined salt concentration was
the optimal elution conditions. This offers a generalized approach
to CEX development work and minimizes development time.
BIOT 5 – 10:10 a.m.
Improved expanded bed adsorption technology for recovery of
biopharmaceuticals
Emile van de Sandt, [email protected], Piet den Boer,
Mark Doeven, Rolf Douwenga.DSM, The Netherlands
The output of cell cultures for biopharmaceutical products has been
improved by mainly advances in molecular biology to increase the
cell line productivity, optimization of media compositions, and
feed delivery strategies. DSM has developed XD® Technology,
able to push the cell density within the bioreactor to the max and
thereby achieve maximum productivity. In the recent years, also
improved separation technologies are developed which are essential to overcome the bottlenecks in downstream processing and to
lower the costs which are currently for a major part in DSP. Improved Expanded Bed Technology, RHOBUST®, has been developed able to improve the recovery process. A rotating fluid device
is able to handle high density cell cultures. Furthermore, increased
density –with tungsten carbide- adsorbent media are available with
protein-A, ion-exchange or mixed mode ligands. Finally, the EBA
technology also comes available in a disposable form.
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Hiromasa Aono1, [email protected], Chanchal
Engineering, University College London, London, United Kingdom (2)
Department of Chemical Engineering, University College London, London,
United Kingdom (3) Pfizer Biopharmaceuticals, Andover, Massachusetts
01810, United States
Randhawa1, Blake R. Pepinsky2, David R.H. Evans1. (1) Department of
Process Biochemistry, Biogen Idec, Cambridge, MA 02142, United States
(2) Department of Protein Biochemistry, Biogen Idec, Cambridge, MA
02142, United States
We demonstrate the application of scanning electron microscopy
(SEM), finite bath uptake experiments, confocal laser scanning microscopy (CLSM) and small scale column studies to understanding
chromatography fouling. Two systems were compared; a hydrophobic interaction capture step for virus-like particles and an anion exchange polishing step following a protein A affinity capture.
In both systems resin samples taken for SEM and finite bath uptake
experiments indicate that as fouling proceeds with successive batch
cycles significant blockage of the pores at the resin surface occurs,
thereby decreasing protein uptake rate. However morphological
differences between the two systems were seen. Further study, conducted using CLSM to allow temporal and spatial measurement
within the resins, was performed using clean, partially fouled and
fully fouled resin samples. Packed within a miniaturized flow cell
the results indicated that the foulant severely decreasing the protein uptake rate, while a minimal decrease in saturation capacity
was found.
Trisulfide has been detected as a source of molecular heterogeneity in protein therapeutics. Chromatography studies using several
different proteins demonstrated that a cysteine (Cys)-containing
wash, performed after loading of the protein and prior to elution,
converted the protein trisulfide bonds to native disulfides. The
Cys wash is a convenient method for trisulfide conversion, and
the studies illustrated the method to be robust under a range of
processing conditions relevant for the manufacturing of protein
therapeutics.
[1] M. Hall and A.S. Bommarius, Chem. Rev. 2011 , 111, 000 (July issue)
[2] A.S. Bommarius, J.K. Blum, M.J. Abrahamson, Curr. Opin. Chem. Biol.
2011,15,194-200
[3] A.S. Bommarius, “Check Nature first, then evolve”, Nature Chem. Biol.
Upstream Processes
Advances in Biocatalysis
8:30 a.m Room# 16B
K. Jones Prather, M. Truppo 2010 , 6, 793-4
Papers 9-16
BIOT 7 – 10:50 a.m.
BIOT 10 – 8:50 a.m.
Biocatalysis scale-up for delivery of chiral intermediates
Tom Moody, [email protected] Sciences, Belfast,
Facilitated protein refolding by like-charged ion-exchangers:
Effects of bead properties
BIOT 9 – 8:30 a.m.
Linling Yu, Xiaoyan Dong, Yan Sun, [email protected]. Department of
Biochemical Engineering, Tianjin University, Tianjin, China
Novel amine dehydrogenases for the production of
chiral amines
Anion exchangers of different ionic capacities, ligand chemistries,
pore sizes and particle sizes were prepared for studying their effects
on the oxidative refolding of like-charged lysozyme. It was found
that ionic capacity of the resins had significant contribution to the
enhancing effects on lysozyme refolding. In the low bead concentration region, the refolding yield increased with increasing ionic
capacity and bead concentration. The refolding yield then reached
a plateau at a critical bead concentration; the higher the ionic capacity, the lower the critical bead concentration. This means that
higher ionic–capacity gel was favorable to offer higher refolding
yield at lower added concentrations. In the bead concentration
range in which refolding yield has reached plateau, there existed an
optimum ionic capacity that gave the highest refolding yield. It was
attributed to the electrostatic screening effect of the charged ionic
groups on the accessible pore volume for the like-charged protein.
ability to create novel enzyme functionality.[2-3]
We have developed two novel amine dehydrogenases (AmDH)
with broad substrate specificity. The AmDH were developed from
existing leucine and phenylalanine dehydrogenase scaffolds. Expansion of its substrate specificity was achieved through several
rounds of focused mutagenesis, allowing for the creation of chiral
amines. This creates a new green route in the production of chiral amines utilizing ketone and NH3 substrates. The AmDH from
phenylalanine dehydrogenase exhibits further enhanced activity
and organic solvent stability over the leucine dehydrogenase-based
enzyme.
Application of degenerate codons and a high-throughput screening assay allowed for simplified, rapid evaluation of enzyme variants. Constraint of mutant library sizes was guided by mechanistic
and structural knowledge, ultimately reducing the screening requirements while maintaining an increased chance of generating
alternate substrate specificity. Novel activity was achieved toward
a number of compounds while maintaining the enzymes’ native
enantioselectivity.
________________
Michael J Abrahamson1, [email protected], Andreas
S Bommarius1, Eduardo Vazquez-Figueroa1, Jeffrey C Moore2, John W
Wong3. (1) Department of Chemical and Biomolecular Engineering,
Georgia Institute of Technology, Atlanta, Georgia 30332, United States (2)
Department of Process Research, Merck Research Laboratories, Merck and
Company, Inc., Rahway, NJ 07065, United States (3) Biocatalysis Center
of Emphasis, PharmaTherapeutics Research & Development, Pfizer Inc.,
Groton, CT 06340, United States
Biocatalysts are increasingly used in industry to create enantiomerically pure compounds; routes employing dehydrogenases
and other redox enzymes have been especially successful.[1] Enantiomeric compounds have a broad range of uses including an
utmost importance in pharmaceuticals. Contemporary methods
of protein engineering, such as applying rational design guided by
mechanistic and structural knowledge, have greatly increased the
United Kingdom
The chemical industry strives to lower costs and the need for economic, robust, scaleable and reliable processes for the synthesis of
chiral APIs and intermediates has resulted in process chemists tuning their skills at the interface of chemistry and biology. Recent
advances in DNA technology have enabled tailoring of enzyme
properties as well as access to increased biocatalyst variety at ever
decreasing cost. This has resulted in the ease of access to “off-theshelf ” biocatalysts that are industrially ready. When developing
manufacturing processes involving biocatalysts, scale up challenges are frequently encountered for both biocatalyst preparation
and chemical transformation. This presentation, through actual
case studies, will illustrate the development of novel biocatalytic
processes for carbonyl-reduction, transamination and biooxidation chemistries. Consideration of scale-up parameters and their
impact on associated costings will be discussed. Recent examples
given include commercially important compounds such as quinuclidinol and woody acetate.
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Sunday Morning
BIOT 4 – 9:30 a.m.
Improving productivity and enzyme stability through process
design: Lipase-catalyzed synthesis of epoxides and esters
Anna E. V. Hagström1,2, [email protected], Mathias Nord-
blad2,3, Ulrika Törnvall2,3, John M Woodley3, Rajni Hatti-Kaul2, Patrick
Adlercreutz2. (1) Department of Chemical and Biomolecular Engineering,
University of Houston, Houston, Texas 77004, United States (2) Department of Biotechnology, Lund University, Lund, Sweden (3) Department of
Chemical and Biochemical Engineering, Technical University of Denmark,
Lyngby, Denmark
Biocatalysis has seen much use in research, but the number of industrial applications is still limited. Part of the reason for this is
the lack of cost-effective processes due to limited stability of the
biocatalyst and productivity in several cases. One of the most important factors in order to achieve an economically feasible process is the operational stability of the enzyme, which is influenced
by the choice of enzyme, catalyst formulation (e.g. immobilization
technique and matrix) as well as the reaction conditions. In this
work the enzyme stability and productivity has been studied for
the synthesis of polyester acrylates, wax esters and epoxidized fatty
acids. All reactions were catalyzed by immobilized lipase preparations. The acrylation reaction proved to be competitive with the
traditional chemical production methods, while the epoxidation
reaction needs further optimization regarding the choice of enzyme and reaction conditions to achieve a viable process.
no improvement in hydrolysis was observed with a third variant
carrying a combination of both mutations, which instead showed
a 60% reduction in catalytic efficiency. This work further demonstrates that non-catalytic amino acid residues can be engineered to
enhance catalytic efficiency in pretreatment enzymes of interest.
operational stability were attained with a directed evolution cutinase mutant T179C, which demonstrated a higher resistance to the
denaturing effect of the anionic surfactant and the alcohol. MBR
could operate for more than 28 days and high productivity levels
(up to 500 gproduct/(day. genzyme)) were achieved.
BIOT 13 – 10:10 a.m.
BIOT 15 – 10:50 a.m.
Artificial cellulosomes for enhanced biomass processing
Structure-function characterization and redox tuning of a
multicopper oxidase for biological fuel cell cathodes
Shen-Long Tsai, [email protected], Qing Sun, Wilfred Chen. Chemical
Engineering, University of Delaware, Newark, DE 19716, United States
Biocatalysis, especially multiple enzyme systems, has been receiving more attention for the production of chemicals such as biofuels.
Cellulosome, a cell-bound multienzyme complex, can be described
as one of nature’s most elaborate and highly efficient biocatalysts
for the deconstruction of cellulose and hemicelluloses. Enzyme assembly occurred via the highly specific cohesin and dockerin interaction, resulting in synergistic biomass deconstruction based on
spatial proximity and enzyme-substrate targeting. However, due to
potential metabolic burdens and protein folding problems, artificial cellulosomes with higher enzyme loadings has not been realized. Our lab has been investigating several different approaches
for the functional assembly of more complex cellulosme structures.
In this talk, we will highlight our recent efforts investigating the
effect of enzyme density, enzyme ordering and enzyme proximity
on the synergism of cellulose hydrolysis and ethanol production.
BIOT 12 – 9:30 a.m.
Novel mutants of the Thermoascus aurantiacus endo-β-1,4
glucanase Eg1 with increased specific activity by rational
mutagenesis
Sneha Srikrishnan1, [email protected], Arlo Zan Randall2, Pierre F.
Baldi2, Nancy A. Da Silva1, 2. (1) Chemical Engineering and Materials
Science, University of California, Irvine, Irvine, CA 92697, United States
(2) Institute for Genomics and Bioinformatics, University of California,
BIOT 14 – 10:30 a.m.
Continuous biodiesel production in a cutinase membrane
bioreactor
Multicopper oxidases (MCOs), such as the small laccase from
Streptomyces coelicolor (SLAC), contain four Cu atoms in the
catalytic site: A type 1 (T1) Cu transfers e- from a reductant to
a trinuclear site to catalyze the 4e- reduction of O2 to H2O. This
unique ability is harnessed in biological fuel cells and SLAC is a
viable cathode catalyst, particularly for operation in conditions
where fungal laccases are not active. Recent molecular dynamic
simulations compared the structure-function relationships between SLAC and other MCOs, which provided insight to mutations
that would influence the T1 Cu redox potential. Herein, we present bioelectrochemical characterization of several SLAC variants.
Alterations in the T1 Cu coordination sphere resulted in changes
in the biochemical properties and the O2 reduction onset potential
of SLAC. Our study represents the potential to tune enzymes for
specific bioelectrocatalytic applications.
Sara M Badenes, [email protected], Francisco Lemos,
Joaquim M S Cabral. Centre for Biological and Chemical Engineering,
Instituto Superior Técnico, IBB - Institute for Biotechnology and Bioengi-
Irvine, Irvine, CA 92697, United States
neering, Lisboa, Portugal
Variants of the Thermoascus aurantiacus Eg1 enzyme with higher
catalytic efficiency than wild-type were obtained via site-directed
mutagenesis. The mutations were chosen based on a rational mutagenesis strategy including extensive family analysis, modeling,
and literature survey. Two promising variants were constructed
and the enzymes were expressed and secreted from Pichia pastoris.
The two mutants showed 1.7- and 4.0- fold increases in kcat with
1.5- and 2.5-fold improvements in hydrolytic activity on cellulosic
substrates, respectively, while maintaining thermostability. Similar
to the parent, the two variants were active between pH 4.0– 8.0 and
showed optimal activity at temperature 70˚C, pH 5.0. In contrast,
Developing alternative energy sources to replace traditional fossil fuels have become a very important task due to the limited
resources of fossil fuel and to environmental concerns. Biodiesel
production by transesterification of oils is an established industrial
process involving inorganic base or acid catalysts. The development of processes that use reusable and environmentally friendly
catalysts is of significant importance. The feasibility of continuous
transesterification of oils in a membrane bioreactor (MBR) by Fusarium solani pisi recombinant cutinase, microencapsulated in sodium bis(2-ethylhexyl) sulfosuccinate/isooctane reversed micelles,
was investigated. The MBR included a ceramic tubular membrane
capable of retaining the enzyme. Promising results of biocatalyst
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D. Matthew Eby1, [email protected], Guinevere M. Strack2,
Lloyd J. Nadeau2, Randi N. Tatum1, Heather R. Luckarift1, Ruth Pachter3,
Glenn R. Johnson2. (1) Universal Technology Corporation, Tyndall Air
Force Base, FL 32403, United States (2) Materials and Manufacturing
Directorate, Air Force Research Laboratory, Tyndall Air Force Base, FL
32403, United States (3) Materials and Manufacturing Directorate, Air
Force Research Laboratory, Wright-Patterson Air Force Base, OH 45433,
United States
BIOT 16 – 11:10 a.m.
Development of a whole-cell biocatalyst for permanent carbon
capture and storage: Immobilization of carbonic anhydrase in
the periplasmic space of Escherichia coli
Tushar Patel1, [email protected], Edward Swanson1, Ah-Hyung
Alissa Park1,2, Scott Banta1. (1) Chemical Engineering, Columbia University, New York, New York 10027, United States (2) Earth and Environmental Engineering, Columbia University, New York, New York 10027, United
States
Carbon mineralization is among the most permanent storage
technologies of anthropogenic CO2. We propose reinventing this
technology to directly fix CO2 in flue-gas as a novel carbon capture process. Carbonate ions from the hydration of CO2 gas and
magnesium ions from mineral dissolution form a thermodynamically stable salt. Due to the slow rate of uncatalyzed-CO2 hydration at near-neutral pH, catalysis would result in a higher rate of
carbon capture. We have developed whole-cell biocatalyst through
periplasmic enzyme immobilization. The motivation behind this
work is to increase the recyclability of the enzyme and decrease
the cost of obtaining the catalyst by eliminating protein purification. Through the use of stopped-flow kinetic measurements, kinetic parameters for the immobilized enzymes have been obtained,
and we have explored the effects of diffusion across the outer cell
membrane of bacterial cells on the catalytic rate, one of the limiting
factors to the feasibility of this system.
Advances in Biofuels Production
Biomass Pretreatment and Hydrolysis
8:30 a.m. Room# 17A
J. Zhu, Y. Kim, M. Ladisch Papers 17-24
BIOT 17 – 8:30 a.m.
Biomass specific pretreatment strategties to improve sugar
recovery while facilitating enzymatic hydrolysis at minimal
enzyme loadings
Richard P Chandra, [email protected], Jack N Saddler Department of Wood Science, University of British Columbia, Vancouver, BC
V6T1Z4, Canada
Nature has designed biomass to resist degradation by enzymes and
pretreatment processes have to be chosen strategically to deal with
this recalcitrance. For acidic pretreatments, such as steam and organosolv, both the xylan component of agricultural and hardwood
biomass and the lignin component of softwood biomass, play a
critical role in influencing the ease of hydrolysis of the resulting
pretreated substrates. To overcome the compromise between maximizing sugar recovery and hydrolysis at minimal enzyme loadings,
different strategies are required. We have shown that the selective
removal of hemicellulose during pretreatment does not facilitate
hydrolysis of softwoods substrates at low enzyme loadings due to
the residual lignin which must be removed via a post treatment.
However, for hardwood and agricultural biomass, one and two
stage pretreatments using various acidic catalysts including SO2
and oxalic acid resulted in the recovery of more than 80% of the
xylan component while maximizing hydrolysis yields at enzyme
loadings as low as 3-7 FPU/g of cellulose.
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Sunday Morning
BIOT 11 – 9:10 a.m.
Lignocellulose pretreatment: Beneficial and nonbeneficial
effects prior enzyme hydrolysis
Eduardo Ximenes, [email protected], Youngmi Kim, Nathan
developing a low cost process for generating sugars from WS will
be discussed.
BIOT 20 – 9:30 a.m.
Mosier, Michael Ladisch.Department of Laboratory of Renewable Resources
Engineering & Agricultural and Biological Engineering, Purdue University,
West Lafayette, IN 47907, United States
Effects of dilute acid pretreatment on cellulose DP and the
relationship between DP reduction and cellulose digestibility
Pretreatment is an important cost-driver of lignocellulose conversion to ethanol and a critical step prior to enzyme hydrolysis. It disrupts the plant cell wall network and partially separates the major
polymer components (lignin, cellulose and hemicellulose). However, pretreatment of lignocellulosic materials may also result in
the release of inhibitors and deactivators of the enzymatic hydrolysis of cellulose. Development of enzyme processes for hydrolysis of
cellulose to glucose must reduce inhibition and deactivation effects
in order to enhance hydrolysis and reduce enzyme usage. Here we
report the identification of phenols with major inhibition and/or
deactivation effect on enzymes used for conversion of cellulose to
ethanol. The strength of the inhibition or deactivation effect depended on the type of enzyme, the microorganism from which the
enzyme was derived, and the type of phenolic compounds present.
The effects of inhibitors on enzyme hydrolysis of pretreated lignocellulosic materials are presented.
E Himmel, David K Johnson.National Renewable Energy Laboratory,
Wei Wang, [email protected], Xiaowen Chen, Melvin Tucker, Michael
Golden, CO 80401, United States
The degree of polymerization(DP) of cellulose is considered to be
one of the most important properties affecting the enzymatic hydrolysis of cellulose. Various pure cellulosic and biomass materials
have been used in a study of the effect of dilute acid treatment on
cellulose DP. A substantial reduction in DP was found for all pure
cellulosic materials studied even at conditions that would be considered relatively mild for pretreatment. The effect of dilute acid
pretreatment on cellulose DP in biomass samples was also investigated. Corn stover pretreated with dilute acid under the most
optimal conditions contained cellulose with a DPw in the range
of 1600~3500, which is much higher than the level-off DP(DPw
150~300) obtained with pure celluloses. The effect of DP reduction
on the saccharification of celluloses was also studied. From this
study it does not appear that cellulose DP is a main factor affecting
cellulose saccharification.
BIOT 19 – 9:10 a.m.
Generation of low cost sugars from wheat straw: Progress,
opportunities, and challenges
Badal C Saha, [email protected], Michael A Cotta. Bioenergy
Research Unit, National Center for Agricultural Utilization Research,
USDA-ARS, Peoria, Illinois 61604, United States
Wheat straw (WS) is a low cost feedstock for production of fuel
ethanol. It contains about 35-45% cellulose, 20-30% hemicelluloses,
and 8-15% lignin. Generation of sugars from WS involves pretreatment and enzymatic saccharification. Pretreatment is crucial as native WS is very resistant to enzymatic hydrolysis. We have studied
in detail hydro-thermal, dilute acid, lime, alkaline peroxide, and
microwave pretreatments prior to enzymatic saccharification using a variety of enzyme preparations. Some of these pretreatments
produce sugar degradation products such as furfural and hydroxymethyl furfural. In this presentation, our research dealing with
these pretreatment options and enzymatic saccharification will be
described. We are able to achieve 82 to 100% conversion of WS to
fermentable sugars. In addition, our research related to development of improved enzymes for lignocellulosic biomass conversion
will be highlighted. The current status, problems, and prospects of
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BIOT 21 – 10:10 a.m.
Redistribution of lignin caused by dilute acid pretreatment
of biomass
David K Johnson1, [email protected], Bryon S Donohoe1, Rui
Katahira2, Melvin P Tucker2, Todd B Vinzant1, Michael E Himmel1. (1)
Biosciences Center, National Renewable Energy Laboratory, Golden, CO
80401, United States (2) National Bioenergy Center, National Renewable
Energy Laboratory, Golden, CO 80401, United States
Research conducted at NREL has shown that lignin undergoes a
phase transition during thermochemical pretreatments conducted
above its glass transition temperature. The lignin coalesces within
the plant cell wall and appears as microscopic droplets on cell surfaces. It is clear that pretreatment causes significant changes in lignin distribution in pretreatments at all scales from small laboratory
reactors to pilot scale reactors. A method for selectively extracting
lignin droplets from the surfaces of pretreated cell walls has allowed us to characterize the chemical nature and molecular weight
distribution of this fraction. The effect of lignin redistribution on
the digestibility of pretreated solids has also been tested. It is clear
that removal of the droplets increases the digestibility of pretreated
corn stover. The improved digestibility could be due to decreased
non-specific binding of enzymes to lignin in the droplets, or because the droplets no longer block access to cellulose.
BIOT 24 – 11:10 a.m.
BIOT 22 – 10:30 a.m.
Juan M Mora-Pale1, [email protected], Hong Wu1, Jianjun Miao1,
Thomas V Doherty5, Luciana Meli1, Robert J Linhardt1,2,3,4, Jonathan S
Dordick1,2,4,5. (1) Department of Chemical and Biological Engineering,
Rensselaer Polytechnic Institute, Troy, NY 12180, United States (2) Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, United
States (3) Department of Chemistry and Chemical Biology, Rensselaer
Polytechnic Institute, Troy, NY 12180, United States (4) Department of
Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180,
United States (5) Department of Materials Science & Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, United States
On sulfite pretreatment to overcome recalcitrance of lignocelluloses (SPORL) for robust bioconversion of woody biomass
Junyong ZHU, [email protected] Products Laboratory, USDA Forest Service, Madison, WI 53726, United States
Wood biomass is a very important feedstock for the future biobased economy. It can be sustainably produced in large quantities
in the United States. However, woody biomass is more recalcitrant
than nonwoody biomass. We have developed the Sulfite Pretreatment to Overcome Recalcitrance of Lignocelluloses (SPORL) process to effectively remove the recalcitrance of woody biomass. We
were able to produce excellent sugar and ethanol yields from both
hardwoods and softwoods at low to moderate enzyme loadings.
The post SPORL pretreatment wood size reduction also significantly reduces energy consumption for wood size reduction, critical to overall energy balance for biofuel production. In this presentation, I will provide a detailed overview of the SPORL process.
The presentation will cover the typical process condition, cellulose
conversion efficiency, mass and energy balance analyses, ethanol
production at high solids loadings. I will also provide comparisons
between SPORL and dilute acid (DA) pretreatment for ethanol
productions.
BIOT 23 – 10:50 a.m.
Preparation of new glycol-functionalized ionic liquids for
biomass pretreatment and hydrolysis
Pretreatment of lignocellulosic biomass at high loadings in
room temperature ionic liquids
Room temperature ionic liquids (RTILs) are emerging as attractive and green solvents for lignocellulosic biomass pretreatment.
We demonstrate that acetate based ILs are effective in dramatically
reducing the recalcitrance of corn stover toward enzymatic polysaccharide hydrolysis even at loadings of biomass as high as 50%
by weight. Under these conditions, the IL serves more as a pretreatment additive rather than a true solvent. Pretreatment of corn
stover with 1-ethyl-3-methylimidizolium acetate ([Emim] [OAc])
at 125 ± 5oC for 1 h resulted in a dramatic reduction of cellulose
crystallinity (up to 52%) and extraction of lignin (up to 44%). Enzymatic hydrolysis of the IL-treated biomass resulted in fermentable sugar yields of ~80% for glucose and ~50% for xylose at corn
stover loadings up to 33% (w/w). Similar results were observed using switchgrass, poplar, and the highly recalcitrant hardwood, maple. At 4.8% (w/w) corn stover, [Emim][OAc] can be readily reused
up to 10 times without regeneration, with no effect on fermentable
sugar yields. A significant reduction in the amount of IL combined
with facile recycling has the potential to enable ILs to be used in
large-scale biomass pretreatment.
Hua Zhao1, [email protected], Gary A. Baker2, John E. Jones1.
(1) Chemistry Program, Savannah State Univeristy, Savannah, GA 31410,
United States (2) Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211, United States
Ionic liquids have gained tremendous attention for the pretreatment of cellulosic biomass in recent years. However, most of these
ionic liquids are based on expensive cations such as imidazoliums,
which makes the ionic liquid pretreatment not cost-effective at
large scales. We have developed new ionic liquids derived from
inexpensive quaternary ammonium or piperidinium salts grafted
with glycol chains. These new ionic liquids are capable of dissolving cellulose, which allows the biomass pretreatment and further
affords an improved enzymatic hydrolysis of lignocelluloses. In addition, these new ionic liquids are considered to be less expensive,
less toxic and more biodegradable.
25
Sunday Morning
BIOT 18 – 8:50 a.m.
Embryonic and Induced Pluripotent
Stem Cells
8:30 a.m.
Room# 25A
R. Rao, W. Grayson Papers 25-31
discovery of novel therapeutics for FXS and other autismspectrum
disorders sharing common pathophysiology.
BIOT 26 – 8:50 a.m.
Guiding the derivation of endothelial cells from human
pluripotent stem cells in a 2D, feeder free differentiation
scheme
Sravanti Kusuma1,2, [email protected], Siah Hong Tan1, Eliza-
BIOT 25 – 8:30 a.m.
Epigenetic characterization of the FMR1 gene and aberrant
neurodevelopment in human induced pluripotent stem cell
models of fragile X syndrome
Steven Sheridan, [email protected] for Human
Genetics, Massachusetts General Hospital/Harvard Medical School, Boston,
MA 02114, United States
Fragile X syndrome (FXS) is the most common inherited cause of
intellectual disability. In addition to cognitive deficits, FXS patients
exhibit hyperactivity, attention deficits, social difficulties, anxiety,
and other autistic-like behaviors. FXS is caused by an expanded
CGG trinucleotide repeat in the 59 untranslated region of the
Fragile X Mental Retardation (FMR1) gene leading to epigenetic
silencing and loss of expression of the Fragile X Mental Retardation
protein (FMRP). Despite the known relationship between FMR1
CGG repeat expansion and FMR1 silencing, the epigenetic modifications observed at the FMR1 locus, and the consequences of the
loss of FMRP on human neurodevelopment and neuronal function remain poorly understood. To address these limitations, we
report on the generation of induced pluripotent stem cell (iPSC)
lines from multiple patients with FXS and the characterization of
their differentiation into post-mitotic neurons and glia. We show
that clones from reprogrammed FXS patient fibroblast lines exhibit
variation with respect to the predominant CGG-repeat length in
the FMR1 gene. In two cases, iPSC clones contained predominant
CGG-repeat lengths shorter than measured in corresponding input population of fibroblasts. In another instance, reprogramming
a mosaic patient having both normal and pre-mutation length
CGG repeats resulted in genetically matched iPSC clonal lines differing in FMR1 promoter CpG methylation and FMRP expression.
Using this panel of patient-specific, FXS iPSC models, we demonstrate aberrant neuronal differentiation from FXS iPSCs that is
directly correlated with epigenetic modification of the FMR1 gene
and a loss of FMRP expression. Overall, these findings provide evidence for a key role for FMRP early in human neurodevelopment
prior to synaptogenesis and have implications for modeling of FXS
using iPSC technology. By revealing disease-associated cellular
phenotypes in human neurons, these iPSC models will aid in the
26
beth Peijnenburg1, Stephen Zhao1, Prashant Mali2,3, Linzhao Cheng3,
Sharon Gerecht1. (1) Chemical and Biomolecular Engineering, Johns
Hopkins University, Baltimore, MD 21218, United States (2) Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21218, United
States (3) Department of Gynecology & Obstetrics and Stem Cell Program,
Institute for Cell Engineering, Johns Hopkins University School of Medicine,
Baltimore, MD 21287, United States
Differentiation of pluripotent stem cells (PSCs) toward endothelial
cells (ECs), which comprise the vasculature’s inner lining, is critical
for the advancement of regenerative medicine. We have developed
highly chemically defined conditions in a clinically-relevant manner for the controlled differentiation and robust derivation of functional ECs from both embryonic and induced pluripotent stem
cells. We examined critical angiogenic growth factors – VEGF,
BMP4, and Ihh – as well as TGFβ inhibitor, SB431542, for their
ability to promote EC differentiation. RT-PCR, immunofluorescence, and flow cytometry analyses revealed that a stepwise process
is required to induce PSC differentiation to ECs. Endothelial derivatives were found to express mature markers including VEcad,
VEGFR2/KDR, and vWF and robustly formed cord-like structures
in vitro. This work has considerable clinical impact with respect
to improved vascular therapeutics and regenerative medicine.
BIOT 27 – 9:10 a.m.
Efficient generation and utilization of epithelial cells derived
from human pluripotent stem cells
Joshua A Selekman1, [email protected], Erin McMillan2, Kyle J
Hewitt3, Jonathan Garlick3, Sean P Palecek1, Joyce Teng2. (1) Department
of Chemical and Biological Engineering, University of Wisconsin-Madison,
Madison, WI 53706, United States (2) Department of Dermatology,
University of Wisconsin-Madison, Madison, WI 53715, United States (3)
Sackler School of Graduate Biomedical Sciences, Tufts University, Boston,
MA 02111, United States
In the past decade, there have been remarkable advances in engineering process to generate functional somatic cell types from human pluripotent stem cells (hPSCs). The next step in the advancing
the production of hPSC-derived cells for biomedical applications
is to develop universal methods to improve efficiency and scalabil-
ity of differentiation strategies, regardless of the somatic cell type
of interest. Toward this goal, we investigated the kinetics of cell
fate determination in various cell subpopulations present in hPSC
specification to epithelial cells to develop a method for optimizing epithelial cell purity and yield by guiding appropriate temporal
presentation of differentiation cues. We then used our epithelial
differentiation strategy to generate epidermal keratinocytes using
induced pluripotent stem cells derived from patients with lamellar
ichthyosis, a genetic skin disease, in order recapitulate the disease
phenotype and study its progression in vitro.
BIOT 28 – 9:30 a.m.
Characterization of extracellular matrix components towards
generation of defined substrates for human pluripotent stem
cell propagation
Raj R Rao, [email protected] of Chemical and Life Science
Engineering, Virginia Commonwealth University, Richmond, Virginia
23284, United States
Challenges exist in the propagation of human pluripotent stem
cells (hPSCs) due to their ability to spontaneously differentiate
and accumulation of chromosomal abnormalities. Recent studies
from our laboratory have shown that extracellular matrix (ECM)
based substrates generated from human fibroblasts successfully
maintained hPSCs in their undifferentiated state for extended periods. We also conducted proteomic analyses to identify the ECM
proteins in mouse embryonic- and two human fibroblast-derived
ECM-based substrates. These studies form the basis for identification of appropriate ECM components to generate defined substrates that synergistically promotes activation of adhesion and
signaling pathways responsible for hPSC self-renewal.
BIOT 29 -10:10 a.m.
What’s in the dish? Characterization of human pluripotent
stem cells with genomics and epigenomics
Jeanne F. Loring, [email protected] Scripps Research Institute,
United States
Human pluripotent stem cells (hPSCs: embryonic and induced
pluripotent stem cells) proliferate indefinitely and can differentiate
into a wide range of diverse cell types. These qualities make hPSCderived cells potentially valuable for in vitro applications, such as
toxicity testing, drug development, and modeling human genetic
disease, and for clinical use in cell replacement therapy. The major
focus for development of hPSC applications is quality control: are
the cells differentiated into the right type of cells? Are the populations homogeneous? Are the cells normal? High information con-
tent genomic and epigenomic analyses have become valuable tools
for diagnosing the phenotypic state of hPSCs, and for testing their
stability during expansion and differentiation. This talk will focus
on the development and use of a very large database, the Stem Cell
Matrix, for genomic and epigenomic analyses.
BIOT 30 – 10:50 a.m.
Spatiotemporal control of human stem cell differentiation
Jerome V Karpiak1, [email protected], Neil Chi1,2, Adah Almutairi1,3,4,5. (1) Department of Biomedical Sciences, University of California at San Diego, La Jolla, CA 92093-0600, United States (2) Department
of Medicine/Cardiology, University of California at San Diego, La Jolla, CA
92093-0613J, United States (3) Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA
92093-0600, United States (4) Department of NanoEngineering, University
of California at San Diego, La Jolla, CA 92093-0600, United States (5)
Materials Science and Engineering Program, University of California at San
Diego, La Jolla, CA 92093-0600, United States
Conventional human embryonic stem cell (hESC) differentiation protocols involve precise temporal addition of instructional
bioactive molecules dissolved in culture medium. The absence of
bulk active nutrient transport throughout three-dimensional embryoid bodies and the characteristic epithelial-like encapsulation
membrane are major barriers to uniform distribution of exogenous
cues. This leads to inefficient differentiation into target mature
cell phenotype. Uncontrolled burst or sustained release delivery
schemes may not be optimal for in vitro differentiation protocols.
Using the H9 cell line (WA09), we developed an hESC culture system that combines rotary suspended tissue culture with embedded
hydrogel delivery vehicles. In parallel we have developed hydrogel
and polymer microspheres that are able to deliver growth factors,
enzymes, and small molecules from within tissue constructs. The
spatiotemporal control that this system offers can be tuned for specific applications in many three dimensional stem cell differentiation systems.
BIOT 31 – 11:10 a.m.
Using label-free screening to investigate stem-cells from their
microanatomical niche
Karthik Balakrishnan1, [email protected], Matthew R Chapman2, Michael J Conboy3, Swomitra K Mohanty1, Eric Jabart3, Haiyan
Huang4, James Hack1, Irina M Conboy3, Lydia L Sohn1. (1) Department
of Mechanical Engineering, University of California, Berkeley, California 94720, United States (2) Biophysics Graduate Group, University of
California, Berkeley, Berkeley, CA 94720, United States (3) Department of
Bioengineering, University of California, Berkeley, CA 94720, United States
(4) Department of Statistics, University of California, Berkeley, CA 94720,
United States
27
Sunday Morning
Stem Cells and Tissue Engineering
Biophysical &
Biomolecular Processes:
Protein Conjugates: From Basic Principles to
Clinically Active Drugs
8:00 a.m.
Room# 25B
C. Meares, P. Senter Papers 32-39
BIOT 32 – 8:00 a.m.
Chemical site-selective protein modification: Development of a
traceless vascular targeting ADC for cancer therapy
Gonçalo J. L. Bernardes1,2, goncalo.bernardes@alumni-oxford.
com, Dario Neri1, Benjamin G. Davis2. (1) Department of Chemistry and
Applied Biosciences, ETH Zurich, Zurich, Switzerland (2) Department of
Chemistry, University of Oxford, Oxford, United Kingdom
Chemical site-selective modification of proteins is a key strategy for
accessing and studying post-translational modifications (PTMs).
Modified proteins have also gained prominence in many therapeutic approaches, including vaccine design and armed antibodies.
Reaction engineering at cysteine: From disulfides to thioethers, a
range of novel reactions and methods that enable controlled, specific modification of proteins at cysteine were developed. These
methods are efficient strategies to access post-translational modifications such as phosphorylation, glycosylation, prenylation, or
acetylation and methylation of lysine residues on proteins in a
controllable, defined manner.Vascular targeting ADC: A novel
strategy for the development of armed antibodies, chemically pro-
28
grammed for the amplified release of a potent cytotoxic drug in
the tumor surroundings will be presented. Site-selective mixed
disulfide formation directly at cysteine yielded an homogeneous,
traceless ADC. When administered intravenously in a syngeneic
immunocompetent model of murine cancer that is not cured by
conventional cytotoxic agents alone, this vascular targeting ADC
potently inhibits tumor growth.
BIOT 33 – 8:25 a.m.
Chemically programmed antibodies and vaccines
Carlos F Barbas, III, [email protected] of Chemistry, Molecular Biology, and the Skaggs Institute for Chemical Biology, The
Scripps Research Institute, La Jolla, CA 92037, United States
Recently, my laboratory has developed a new class of immunotherapeutic agents termed Chemically Programmed Antibodies or
CovX-bodies as prepared by Pfizer-CovX Inc. In this presentation,
I will attempt to summarize some of our results concerning this
promising new class of immunotherapeutic molecules. In the later
part of my talk I will consider a new approach to vaccines based a
chemical approach to vaccinology that has the potential to provide
‘instant immunity’. Given time, new conjugation chemistries will
be considered.
BIOT 34 – 8:50 a.m.
Chemoselective strategies for the synthesis of proteins and
labeling of nanoparticles
Philip E. Dawson, [email protected] of Chemistry,
The Scripps Research Institute, La Jolla, CA 92037, United States
Chemical ligation approaches have become essential tools for the
engineering of complex molecules including proteins, nucleic acids
and nanoparticles. What makes these reactions so useful is their
compatibility with the biological “solvent” water, and a high level
of chemoselectivity that enables their application in complex molecular environments. We have worked to develop several ligation
chemistries that are highly chemoselective and have sufficient ligation rates to be useful at low concentrations. In one case, the use
of hydrolysis resistant thioester peptides that undergo inter- and
intramolecular acyl transfer enables the total synthesis of proteins.
The optimization of the ligation methodology, improved routes to
the required peptide intermediates, and application of these methods to complex targets will be presented. Another challenge is the
covalent assembly of macromolecules and nanoparticles. In these
systems, a “native” linkage is irrelevant and the main criteria for
a successful ligation methodology are fast reaction rates and high
chemoselectivity. We have found that aniline catalyzed hydrazone
and oxime reactions enable the controlled assembly and disassembly of macromolecular complexes in aqueous solution at micromolar concentrations. The scope of these reactions and new approaches for their catalysis will be discussed.
BIOT 35 – 9:15 a.m.
Permanent and cleavable linkages for protein therapeutics
M.G. Finn, [email protected], Cody Higginson, Alexander Kislukhin,
Srinivas Chirapu.Department of Chemistry, The Scripps Research Institute,
La Jolla, CA 92037, United States
The chemical modification of proteins requires highly active and
selective reactions of the “click chemistry” variety. For certain applications, the triggered release of cargo molecules is also advantageous. The latest developments in the copper-catalyzed azidealkyne cycloaddition reaction for bioconjugation will be described,
along with the attachment and release reactions of electron-deficient oxanorbornadiene electrophiles for thiol addition.
BIOT 36 – 9:50 a.m.
Photo-immunotherapy: A super-controlled molecular targetspecific cancer theranostics, evolving from the molecular
imaging technology
Hisataka Kobayashi, [email protected], Makoto Mitsunaga,
Mikako Ogawa, Peter L Choyke.Molecular Imaging Program, NCI/NIH,
Bethesda, MD 20892-1088, United States
Three modes of cancer therapy, surgery, radiation and chemotherapy, have been central to modern oncologic therapy. Here, we
employ an activatable hydrophilic photosensitizer based on a near
infrared (NIR) phthalocyanine, IR700, which is covalently conjugated to one of several humanized monoclonal antibodies (MAb)
targeting cancer-specific cell-surface molecules. When exposed to
NIR light, the conjugate induces highly selective cancer cell death
both in vitro and in vivo, a process termed “photo-immunotherapy” (PIT). Additionally, IR700 fluorescence produced by the MAbconjugate permitted identification of the target, image-guided light
exposure, and tumor monitoring after therapy. The MAb-IR700
PIT was effective when conjugates were bound to the cell membrane even at 4°C, but showed no phototoxicity, when unbound,
suggesting a novel mechanism for cytotoxicity compared with conventional photodynamic therapies. In conclusion, this theranostic
image-guided target-selective PIT based on MAb-IR700 cell membrane binding enables super-selective treatment of cancer cells
without apparent side effects to surrounding tissue.
BIOT 37 – 10:15 a.m.
Toward specific covalent targeting of cancer
Heather E. Beck, Bernadette V. Marquez, Claude F. Meares,
[email protected] of Chemistry, University of California,
Davis, California 95616, United States
Delivering probes to cancer cells in vivo for imaging or therapy is
best accomplished with technology that [1] specifically accumulates probe molecules on the target cells, wherever they may be;
[2] delivers sufficient probe concentration and time of residence in
the target; [3] does not leave probes in normal tissue; and [4] clears
untargeted probes efficiently from the body. Antibody technology
generally meets criteria [1] and [2] very well, while peptides and
other synthetic ligands can do better with the other criteria in some
cases, and nanoparticles offer promise for the future. The most
compelling practical challenges to be addressed involve specificity
and duration of target binding, and transport properties of the signal-bearing probe molecules. Our lab has devised a strategy based
on a combination of protein engineering and synthetic chemistry
that allows us to deal separately with these challenges. Supported
by NIH research grants CA0168961, CA136639.
BIOT 38 – 10:40 a.m.
Antibody-conjugated immunopolymeric nanoparticles for
targeted chemotherapeutic delivery
Molly S Shoichet1,2,3, [email protected], Jiao Lu3, Karyn
Ho1,2, Shawn Owen1,2, Dianna Chan1,2, Molly K Gregas1,2, Jennifer Logie1,2, Ira Schmid3, Dev Sidhu4, Nish Patel4. (1) Chemical Engineering
& Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5,
Canada (2) Institute of Biomaterials & Biomedical Engineering, University
of Toronto, Toronto, Ontario M5S 3G9, Canada (3) Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada (4) Banting
and Best Department of Medical Research, University of Toronto, Toronto,
Ontario M5G 1L6, Canada
Selective drug delivery to solid cancer tissues remains a major
challenge in chemotherapeutic treatment strategies. To overcome
this challenge, drug-loaded polymeric nanoparticles have been
designed to take advantage of the enhanced permeability and retention effect associated with tumour vasculature, which should
result in their specific accumulation in tumour tissue. To test this
hypothesis, we synthesized a series of biodegradable amphiphilic
polymers that self-assemble to nanoparticle micelles comprised of
poly(D,L-lactide-co-2-methyl-2-carboxytrimethylene carbonate)g-poly(ethylene oxide)-furan. These nanoparticles can encapsulate small molecule hydrophobic drugs, have low critical micelle
concentration, and are stable in blood serum proteins in vitro and
accumulate in tumours in vivo using an orthotopic breast cancer
29
Sunday Morning
We have developed a label-free cell-analysis platform to characterize key cell-surface markers in functional organ stem cells isolated from their micro-anatomical niche. Our platform measures
the transit time of single cells as they travel through a microchannel functionalized with a specific antibody. Cells expressing the
complementary surface antigen experience a longer transit time
compared to those that do not. We have discovered that individual myofibers differ from each other with respect to expression of
the markers Sca-1, CXCR4, β1-integrin, and M-cadherin on their
associated satellite cells. Our data highlights the phenomenon of
microniche-dependent variation in adult stem cells of the same tissue. Overall, our method can be broadly applied to the quantitative analysis of single stem cells isolated from their microniches in
other adult and developing organs, leading to new discoveries on
stem-cell properties and regenerative potential.
mouse model. Our polymeric micelles, modified with targeting
antibodies via Diels-Alder chemistry, showed selective cytotoxicity
towards cancer cells in vitro and thereby demonstrate cancer cell
targeting. These data demonstrate the utility of this nanoparticle
platform for tumour targeting.
BIOT 39 – 11:05 a.m.
Development of a potent antibody drug conjugate for the
clinical treatment of CD-30 positive malignancies
Peter Senter, [email protected], Seattle Genetics, Bothell,
WA 98021, United States
Monoclonal antibodies (mAbs) play significant roles in the chemotherapeutic treatment of cancer. In an effort to extend their therapeutic potential, we have developed novel technologies surrounding antibody-drug conjugates (ADCs). The goal of this work is to
utilize mAbs for the specific delivery of highly cytotoxic drugs to
cancer cells, while sparing normal tissues from chemotherapeutic
damage. The key parameters in developing an active and well tolerated ADC are the selectivity profiles of the mAb, the potency and
stability of the cytotoxic payload, the conditional stability of the
linker used to attach the drug to the mAb, and the technology used
to control the site and stoichiometry of drug attachment. This presentation will overview how all of these issues were taken into account, leading to the development of ADCETRIS (SGN-35, brentuximab vedotin), a potent ADC that is now clinically approved
for the treatment of Hodgkin lymphoma and anaplastic large cell
lymphoma.
Sunday Afternoon
Sessions
Sunday Afternoon Sessions
A
B
C
Room# 16A
Downstream Processes: Advances in Chromatographic Separations New Materials
2:00 p.m D
E
30
2:00 p.m.
A. Lenhoff, C. Gillespie, K. Eriksson Papers 40-47
Room# 16B
Upstream Processes: Advances in Systems Biology
I.Famili, G. Panagiotou Papers 48-55
2:00 p.m. Room# 17A
Advances in Biofuels Production: Photobiology and Non-traditional Feedstocks
M. Lipscomb, J. Zhu, Y.Kim Papers 56-63
2:00 p.m.
Room# 25A Stem Cells and Tissue Engineering: Adult Stem Cells
B. Harley, S. Palecek Papers 64-70
2:00 p.m. Room# 25B
Biophysical & Biomolecular Processes:
Proteins at Interfaces: Manufacturing, Formulation, and Delivery
J. Champion, Y. Gokarn Papers 71-78
6:30 - 8:30 p.m.
675 L St. at the Omni Hotel
Offsite Reception BIOT Members Only
McCormick & Schmick’s
31
Advances in Chromatographic Separations:
New Materials
2:00 p.m.
Room# 16A
A. Lenhoff , C. Gillespie, K. Eriksson Papers 1-8
BIOT 40 – 2:00 p.m.
Improved bi-layered bi-functional chromatography matrices
Owen R.T. Thomas1, [email protected], Eirini Theodosiou1,3, Kritsandanchalee Karnchanasri1, Thomas C. Willett1, James L.
Walsh2. (1) School of Chemical Engineering, The University of Birmingham, Edgbaston, Birmingham, West Midlands B15 2TT, United Kingdom
(2) Department of Electrical Engineering and Electronics, University of
Liverpool, Liverpool, United Kingdom (3) Department of Chemical Engineering, Loughborough University, Leicestershire, United Kingdom
The potential benefits chromatographic media featuring two distinct functional regions spatially separated from one another within the same support bead have been clearly demonstrated in the
context of ‘nanoplex’ purification, and fluidised bed separation of
organic acids and proteins. However, all bi-layered supports made
thus far, fall short of the ideal design brief of a thin inert outer
size excluding layer, freely accessible to smaller components, but
not larger entities, surmounting a fully functionalised adsorptive
core. In this presentation, we describe our recent progress on the
development of improved bi-layered, bi-functional supports. Specifically we report on the use of: ‘bottom-up’ microwave-assisted
and viscosity enhanced ‘reaction-diffusion balancing’ techniques,
which permit different functionalities to be built into underivatized SEC matrices as discrete ‘onion-like’ layers; and ‘top-down’
plasma methods, which allow one to selectively modify the exteriors of commercial adsorbents, either by shaving ligands away and/
or burying them beneath a thin polymer graft.
BIOT 41 – 2:20 p.m.
Layered beads: A new purification tool in downstream
processing of biomolecules
Tobias Söderman, [email protected] & D, GE Healthcare,
Life Sciences, Uppsala, Sweden
Layered beads is a completely new design concept for bioprocess
chromatography media. The technology is based on beads func-
32
tionalized in layers that gives the beads interesting chromatographic properties and new features compared to traditional chromatography media. One example of a layered bead is called Core Beads,
where ligands are situated exclusively in the interior of the bead
without any functionality in the outer layer. This design enables
the bead to have a molecular weight cutoff determined by the bead
pore size and a functionality determined by the ligand in the core.
This type of media is interesting for flowthrough purification of
large molecules such as viruses and plasmid DNA.
Another type of layered bead is called Shell Beads. In this case the
ligands are situated exclusively in the outer shell of the particles.
This leads to highly improved mass transfer and enables higher
resolution than traditional chromatography media with the same
particle size. Shell Beads are an interesting alternative for any polishing step. The design concept of layered beads will be presented
along with application examples that illustrate the potential uses
of layered beads.
BIOT 42 – 2:40 p.m.
Strategies for enhancing binding capacity and alkaline
stability of peptide-based affinity adsorbents
Stefano Menegatti2, [email protected], Amith D. Naik2,
Ruben G. Carbonell1,2. (1) Biomanufacturing Training and Education
Center (BTEC), North Carolina State University, Raleigh, North Carolina
27695, United States (2) Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695,
United States
Our group has developed peptide affinity ligands HWRGWV,
HYFKFD and HFRRHL for the purification of antibodies. Conventionally, peptide-based adsorbents are produced by direct onresin peptide synthesis by Fmoc/tBu coupling chemistry. This
procedure, however, has two main drawbacks, (1) the formation
of truncated peptide sequences, and (2) the coupling of a significant portion of peptides to the matrix via alkaline labile bonds. The
former lowers the adsorbent capacity and specificity, while the latter results in massive ligand leaching during alkaline cleaning and
sanitization. Therefore, we have implemented several strategies for
the immobilization of pure peptide on chromatographic resins to
optimize binding capacity and selectivity. This work presents different chemistries used for the coupling of pure peptide ligand on
chromatographic resins. Furthermore, to address the problem of
alkaline lability we have developed a method of resin surface modification that allows the peptide coupling to the resin surface exclusively by alkaline-stable bonds.
BIOT 43 – 3:00 p.m.
Hyperthermophilic affinity ligand for virus purification by
avidity chromatography
Mahmud Hussain1, Dustin M Lockney2, Ruqi Wang2, Nimish Gera1,
Balaji M Rao1, [email protected]. (1) Department of Chemical and
Biomolecular Engineering, North Carolina State University, Raleigh, Nc,
United States (2) Department of Chemistry, North Carolina State University, Raleigh, NC, United States
We have generated a stable (Tm ~ 83 deg.C) binding protein for
Red clover necrotic mosaic virus (RCNMV), a plant virus, through
mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus. The Sso7d-based RCNMV-binding
protein (RBP) was then used to purify RCNMV from plant sap, using a variation of affinity chromatography. In this scheme – termed
avidity chromatography – RCNMV from plant sap is captured
on a nickel column that is pre-loaded with hexahistidine tagged
RBP. The highly avid interaction between RCNMV and immobilized RBP ensures efficient capture of RCNMV despite modest
binding affinity (KD ~ 100 nM) of the RBP-RCNMV interaction.
Subsequently, RBP is eluted from the column along with RCNMV;
a single density gradient ultracentrifugation step is used to separate the RBP from RCNMV, as well as concentrate pure RCNMV.
This purification scheme precludes the need for harsh elution conditions that are typically required for affinity chromatography of
viruses and also eliminates the need for chemical conjugation of
the affinity ligand to a resin. Thus, Sso7d based binding proteins
are well-suited for use as affinity ligands for viruses in general, and
may reduce the cost and complexity of virus purification.
BIOT 44 – 3:40 p.m.
Next generation anion exchange membrane adsorbers for
downstream processing
Nathalie Fraud1, [email protected], Kevin Shomglin2, Amit Mehta2, Louis Villain1, Martin Leuthold1, Rene Faber1. (1)
R&D, Sartorius Stedim Biotech GmbH, Goettingen, Germany (2) Purification Process Development, Genentech Inc., South San Francisco, CA 94080,
United States
In anion exchange flow-through (FT-AIEX) applications membrane chromatography offers several advantages over traditional
bead columns. Conventional quaternary amine based chemistries
require typically low feed conductivity and involves often dilution
of feed streams, which can result in facility fit limitations when high
titer processes are accommodated in existing plants. To address
these limitations and facilitate a wider design space for FT-AIEX
membrane chromatography at commercial scale, a new generation
of positively charged salt tolerant membrane adsorbers (Sartobind
STIC®) was developed. Additionally, an ultrascale down device
was developed to minimize the feedstock requirements for process
development, characterization and validation. In this study, the
performance of Sartobind STIC® was explored for host cell protein
clearance using industrially relevant MAb feedstreams at different
experimental conditions. The membrane adsorber was also tested
for clearance of model viruses spiked in MAb feedstreams. Data
demonstrating the scalability of ultrascale down device to process
scale will also be presented.
BIOT 45 – 4:00 p.m.
Optimal feed-specific operating conditions for impurity and
virus removal for a membrane adsorber polishing step
Blaine M Trafton, [email protected], Benjamin Roman,
Michael J Felo.Biomanufacturing Sciences Network, EMD Millipore, Billerica, MA 01821, United States
The use of membrane adsorbers for the removal of trace impurities during biopharmaceuticals production continues to garner
interest and evaluation for clinical and commercial processes.
The robustness of this technology’s performance across various
expression systems, molecules types, and process and operating
conditions remains a key concern for the adoption and implementation of membrane absorbers for large scale manufacturing.
For a commercially available anion-exchange membrane adsorber, the impact of feed conditions and molecule properties on the
removal of impurities such as host cell protein, DNA, virus, and
endotoxin are examined based on a number of studies and products. From this series of studies, feed-specific operating windows
are evaluated and recommended to maximize overall impurity and
virus clearance and product loading. The levels of impurity and virus removal achieved were in line with expected performance for
a polishing step.
BIOT 46 – 4:20 p.m.
Interplay between ionic and hydrophobic interactions in
protein chromatography on multi-modal stationary phases
Michael S. Schmidt2, Heiner Graalfs1, Christian Frech2, c.frech@
hs-mannheim.de. (1) Merck KGaA Department of Research and Development: Chromatography Media, Merck Millipore, Darmstadt, Germany (2)
Hochschule Mannheim: Department of Biochemistry, University of Applied
Sciences Mannheim, Mannheim, Germany
Six new multi-modal strong cation exchangers with varying fractions of hydrophobic groups were synthesized. The prototype materials belong to the class of polymer coated stationary phases,
which carry the functional groups on long flexible tentacles. Based
33
Sunday Afternoon
Downstream Processes
BIOT 47 – 4:40 p.m.
Novel affinity platforms for the purification of r/n proteins
and antibody fragments
Henrik Ihre, [email protected] of Custom Designed
Media, GE Healthcare, Uppsala, N/A 75184, Sweden
The Custom Designed Media department within GE has developed
new BioProcess affinity resins for the purification of e.g. Kappa and
Lambda Fab’s but also for well-established r/n plasma proteins such
as Factor VII, Factor VIII, Alpha-1 Antitrypsin and even affinity
purification of Adeno associated viruses all in close collaboration
with specific customers allowing for purities >95% in one step and
in high recoveries. The development work behind these resins will
be described in more detail as well as some of the other potentials
that these resins also offer. Considering that this is the first generation of affinity resins time will also be spent on discussions on how
the next generation of these resins may be improved to meet future
needs from the industry.
Upstream Processes:
BIOT 48 – 2:00 p.m.
sion. This novel systems biology approach is especially useful for
multifunctional proteins. Specifically, we develop an approach
termed “graded dominant mutants” in which a dominant allele is
used to competitively inhibit and grade a specific protein function.
Using the yeast histone acetyltransferase Gcn5p as a case study, we
demonstrate the utility of this approach by identifying previously
unknown gene targets and interactions and implicating a strong
role for Gcn5p-acetylation in global gene repression. Our results
demonstrate that traditional genetic approaches can significantly
misrepresent the number and identity of gene interactions and
phenotypes.
Evaluating metabolic network elements via a forced
coupling algorithm
BIOT 50 – 2:40 p.m.
Advances in Systems Biology
2:00 p.m Room# 16B
I.Famili, G. Panagiotou Papers 48-55
Christopher J Tervo, Jennifer L Reed, [email protected]
of Chemical and Biological Engineering, University of Wisconsin-Madison,
United States
Genome-scale models allow interrogation of microbial
reaction networks for a variety of purposes such as guiding metabolic engineering strategies and facilitating hypothesis-driven discovery. The success of in silico models depends on their ability to
capture the underlying characteristics of the microbe of interest. To
facilitate model testing and validation, we have developed a forced
coupling algorithm (FOCAL) that can design experiments to test
for the existence of reactions or gene to reaction associtions. FOCAL proposes mutant strains and media conditions under which
a particular network element is conditionally essential for cell
growth or coupled to another measurable reaction (e.g. chemical
production). By testing such conditions experimentally, hypothesized network elements can be confirmed or refuted and models
can be subsequently refined. We will present FOCAL results for
three different genome scale models and illustrate how it can be
used for a variety of applications (model testing, experimental design, and metabolic engineering).
BIOT 49 – 2:20 p.m.
Linking genotype and phenotype through a graded dominant
mutant approach
Hal S. Alper, [email protected], Amanda Lanza, John Blazeck,
Nathan Crook.Department of Chemical Engineering, The University of
Texas at Austin, Austin, TX 78712, United States
Systems biology seeks to establish a relationship between genotype
and phenotype. However, commonly invoked approaches for probing this regulation (such as gene deletions) do not accurately link
protein function and phenotype in a causative manner. Here we
describe a novel approach to extract quantitative, causative links
between specific protein function and downstream gene expres-
34
Comprehensive reconstruction of E. coli metabolism and
application to model-driven metabolic engineering and omics
data analysis
Adam M. Feist, [email protected], Bernhard O. Palsson.Department of
Bioengineering, University of California, San Diego, La Jolla, CA 92093,
United States
The initial genome-scale reconstruction of the metabolic network
of Escherichia coli K-12 MG1655 was assembled in 2000. It has
been updated and periodically released since then based on new
and curated genomic and biochemical knowledge. An update
has now recently been built, named iJO1366, which accounts for
1366 genes, 2251 metabolic reactions, and 1136 unique metabolites. Like its predecessors, the iJO1366 reconstruction is expected
to be widely deployed for studying the systems biology of E. coli
and for metabolic engineering applications. The procedures and
approaches used to update the reconstruction in the era of omics
data sets will be presented. Further, the use of the reconstruction
towards guided design of networks for metabolic engineering purposes through simulation and integration with various omics data
types will be discussed.
BIOT 51 – 3:00 p.m.
Contrasting the metabolic capabilities of cyanobacterial
species for assessing bio-production platform selection
Rajib Saha1, [email protected], Bertram M. Berla2, Thomas Mueller1,
Thanura Elvitigala3, Lawrence E. Page3, Himadri B. Pakrasi3, Costas D.
Maranas1. (1) Department of Chemical Engineering, The Pennsylvania
State University, University Park, PA 16802, United States (2) Department of Energy, Environmental, and Chemical Engineering, Washington
University in St. Louis, St. Louis, MO 63130, United States (3) Department
of Biology, Washington University in St. Louis, St. Louis, MO 63130,
United States
bust growth under diverse environmental conditions with minimal nutritional requirements. They can use solar energy to convert
CO2 and other reduced carbon sources into a variety of biofuels
and chemical products. Here we reconstruct and subsequently analyze and contrast the differences in metabolism between a number
of cyanobacteria species along with their suitability as microbial
production platforms. Specifically, we compare seven genomescale models for two phylogenetically related cyanobacterial species, namely Cyanothece (51142, 7424, 7425, 7822, 8801 and 8802)
and Synechocystis 6803. Reconstructed genome-scale metabolic
models include experimentally-verified biomass compositions,
fully-traced photosynthesis reactions and respiratory chains as
well as balanced reaction entries and GPR associations. We describe results from comparing important pathways leading to biofuel or other useful product formation and subsequently explore
individual potential of these strains as bio-production platforms
and experimental feedback.
BIOT 52 – 3:40 p.m.
Balancing act in the endoplasmic reticulum: Relative
folding and disulfide bond formation rates are key to secretory
homeostasis
Keith EJ Tyo1, [email protected], Zihe Liu2, Dina Petranovic2,
Jens Nielsen2. (1) Department of Chemical and Biological Engineering,
Northwestern University, Evanston, IL 60201, United States (2) Department of Chemical and Biological Engineering, Chalmers University of
Technology, Goteborg, Sweden
In Saccharomyces cerevisiae, recombinant protein secretion can
be plagued with low titers. While different genetic or bioprocessing modifications may improve titer for a particular protein, these
strategies are not generalizable to other proteins. This implies there
are several possible bottlenecks in the secretory pathway, and producing different proteins may be constrained in different ways.
In this study, we use a system level analysis (transcriptomics, metabolomics, and physiological responses) to characterize S. cerevisiae secreting different size proteins, while perturbing secretory
homeostasis through HAC1. Prominent changes in redox stress
and NADPH consumption lead us to develop a redox model for
disulfide bond formation in the ER. By accounting for electron affinities between redox partners, we could predict futile cycles that
produce ROS and could propose hypotheses to mitigate the futile
cycle. This work identifies counterintuitive interventions (slowing
disulfide bond formation or protein synthesis) that may improve
recombinant protein production more generally.
Cyanobacteria are photoautotrophic prokaryotes that exhibit ro-
35
Sunday Afternoon
on the same polymer chemistry an increasing number of hydrophobic groups were incorporated leading to prototypes with ~10
to 30% of identical hydrophobic groups.
Isocratic as well as gradient elution data of monoclonal antibodies
and their fragments at different pH values using different salts are
used to evaluate the influence of ionic as well as hydrophobic interactions on protein retention. Retention models for ion exchange
and hydrophobic interaction chromatography based on the work
of Mollerup and Melander are used to calculate their model parameters. As the prototype stationary phases are all based on the
same base matrix and same surface modification chemistry a direct
correlation of the model parameter to stationary phase properties
is possible.
A good agreement between isocratic retention data and gradient
results is obtained. The contribution to retention by electrostatic
as well as hydrophobic interactions can separately be determined.
The ionic strength range in which both contributions are relevant
is precisely predicted. Additional data on Gibbs energy of adsorption, number of electrostatic interactions, released water molecules
upon binding etc. give an almost complete picture of the basic
principles underlying the chromatographic process. Due to the
systematic variation of stationary phase properties the results are
of direct relevance for the design of new chromatographic media.
Using functional metagenomics to dissect the dissemination of
antibiotic resistance genes
Morten Sommer, [email protected] of Systems Biology,
DTU, Lyngby, DenmarkNovo Nordisk Foundation Center for Biosustainability, Hørsholm, Denmark
The increasing levels of multi-drug resistance in human pathogenic bacteria are compromising our ability to treat infectious disease. Since antibiotic resistance determinants, often encoded on
mobilizable elements, can be readily transferred between bacteria,
we must understand the relative abundance and diversity of reservoirs of resistance genes encoded within microbial communities
from different environments and their accessibility to clinically
relevant pathogens. We have applied experimental metagenomic
approaches to identify and characterize antibiotic resistance genes
from diverse environments including the human microbiome. We
find that some of these genes are identical or highly similar to resistance genes from many clinically relevant multi-drug resistant
human pathogens. Based on these results a framework is emerging
that describes how antibiotic resistance genes are exchanged between microbial communities and which resistance reservoirs are
most accessible to pathogens.
BIOT 54 – 4:20 p.m.
Systematic identification of condition-specific regulatory
module with a novel multi-layer inference approach
Ming Wu1, [email protected], Christina Chan1,2,3, krischan@egr.
msu.edu. (1) Department of Computer Science and Engineering, Michigan State University, East Lansing, Michigan 48824, United States (2)
Department of Chemical Engineering, Michigan State University, East
Lansing, Michigan 48824, United States (3) Department of Biochemistry
and Molecular Biology, Michigan State University, East Lansing, Michigan
48824, United States
The advent of high-throughput data has enabled the global analysis
of the transcriptome, driving the development and application of
computational approaches to study gene regulation on the genome
scale, by reconstructing in silico the regulatory interactions of the
gene network. Thus far, the tools for network inference have been
predominantly based on statistical dependencies between gene expressions. These methods have been shown to capture only part of
the relevant biological information, and usually generate too many
candidates/hypotheses to be experimentally assessed, making the
biological interpretation of the inferred network difficult. We aim
to address these challenges and propose a novel multi-layer network inference approach which integrates gene expression data
and transcriptional regulation information to identify specific
36
regulatory modules for a given condition. Our reverse engineering
framework consists of three layers. The first layer integrates microarray data from a diverse set of conditions to provide a common context of gene behaviors and then identifies the most specific
candidate genes for a given condition. In the second layer we apply
a state-of-art causal filtering method that combines information
on the protein-interaction and protein-DNA interaction to reconstruct the regulatory pathway to the candidate genes. In the third
layer we develop a novel method of inferring transcription factor
activity to identify the important regulators in the gene network
that can account for post-transcriptional regulation. By integrating multiple layers of learning, our framework captures different
biological features in the transcriptional regulation to achieve an
accurate reconstruction of condition-specific gene networks. We
established the accuracy of our methodology against a synthetic
dataset as well as the yeast dataset, and finally applied the approach
to identify novel targets and regulatory mechanisms for human
breast cancer.
growth and branching rates in complete minimal medium, implying autophagy may be active even in the absence of starvation. In
addition, the Anatg13 deletion appears to impact morphology as
these mutants show no significant change in branching rate in any
of the various growth environments employed. This implies autophagy, or the AnAtg13 protein, may play a role in regulation of
fungal branching. We will discuss these findings and their potential
application in the bioprocess industry.
Advances in Biofuels Production:
Photobiology and Non-traditional Feedstocks
2:00 p.m. Room# 17A
M. Lipscomb, J. Zhu, Y.Kim Papers 56-63
BIOT 57 – 2:20 p.m.
BIOT 55 – 4:40 p.m.
Systems biology of fungal autophagy
Mark R. Marten1, [email protected], Bill Moss1, Nikhil Ramsubramaniam1, Usha Sripathineni1, Mariana Silva1, Chris Yankaskas1, Brian
Kirsch1, Cheyenne Falat1, Colleen Courtney1, LaTonya Simon1, Steven
Harris2. (1) Chemical Biochemical and Environmental Engineering,
UMBC, Baltimore, MD 21250, United States (2) Plant Science Initiative,
University of Nebraska, Lincoln, Lincoln, NE 68588, United States
While pathogenic fungi are responsible for numerous deaths and
billions of dollars in crop damage each year, fungi used in the
bioprocessing industry are used to produce billions of dollars in
beneficial products. In nearly all relevant environments, fungi
are likely to experience some degree of starvation, where survival requires recycling internal components in a process called
autophagy. Yet, in spite of it’s likely importance, relatively little
information is available on autophagy, or its related effects, in filamentous fungi. We have systematically deleted a number of putative autophagy genes from the model fungus Aspergillus nidulans,
and are using a sophisticated set of experimental tools to assess
subsequent phenotypes providing insight regarding gene function.
Surprisingly, autophagy appears to be involved in regulation of a
diverse set of, seemingly, unrelated cellular processes. For example,
proteomic analysis shows that deletion of a putative autophagy
gene (Anatg13) leads to altered expression of a number of proteins
related to chronological life span (CLS). Shake flask experiments
on this strain show that low levels of autophagy (induced through
addition of rapamycin) lead to increased CLS, but this phenotype
is abolished in Anatg13 deletion strains. Autophagy also appears
to impact fungal cell walls, and fungal morphology. For example,
Anatg13 deletion mutants show a significant reduction in both
modeling technique on an electrosynthetic bacterium, Clostridium
ljungdahlii, to characterize this process for autotrophic synthesis of
multi-carbon organic compounds such as butanol. We have reconstructed the genome-scale metabolic network of this electrosynthetic organism. This reconstruction comprises of 675 metabolic
reactions encoded by 618 genes, and captures all the major central
metabolic, biosynthetic and energy conservation pathways. Importantly, this network represents one of the first detailed descriptions
of key electrosynthesis pathways. The genome-scale model is used
in conjunction with physiological data to extensively characterize
the various metabolic phenotypes of C. ljungdahlii. We further
employed in silico strain-design tools on the validated metabolic
model to optimize butanol production under electrosynthetic conditions. Here we present the first metabolic network of a homoacetogen and its application as a strain-design platform for optimizing
microbial electrosynthesis.
Enhancement of CO2 and H2 uptake for the production of
biodiesel in Cupriavidus necator
BIOT 56 – 2:00 p.m.
Genome-scale modeling of microbial electrosynthesis for
electrofuel production
Karsten Zengler1, [email protected], Harish Nagarajan1, Juan
Nogales1, Merve Sahin1, Ali Ebrahim1, Derek R. Lovley2, Bernhard O.
Palsson1, Adam M. Feist1. (1) Department of Bioengineering, University
of California San Diego, La Jolla, California 92093, United States. (2) Department of Microbiology, University of Massachusetts Amherst, Amherst,
Massachusetts 01003, United States
A novel mechanism, known as microbial electrosynthesis, in which
microorganisms directly use electric current to reduce carbon dioxide to multi-carbon organic compounds that are excreted from
the cells into extracellular medium, has recently been discovered.
Microbial electrosynthesis differs significantly from photosynthesis in that carbon and electron flow is primarily directed to the
formation of extracellular products, rather than biomass. However,
extensive knowledge about the metabolism of the organism as well
as its extracellular electron transfer pathways is critical to realize
the potential of this technology for the production of the desired
fuel compound. So far, only a few acetogens have been shown to be
capable of accepting electrons from the cathode to reduce carbon
dioxide to limited organic compounds such as acetate and 2-oxobutyrate. Constraint-based metabolic modeling and analysis has
been useful for discovering and understanding new capabilities
and content in bacteria, as well as in guiding metabolic engineering efforts for targeted production.
In this study, we present the application of this constraint-based
Ryan P Sullivan, [email protected], Carrie A Eckert, Grant J Balzer, Jianping Yu, Pin-Ching Maness.Biosciences Center, National Renewable
Energy Laboratory, Golden, CO 80401, United States
Cupriavidus necator fixes CO2 through the Calvin-BensonBassham (CBB) cycle using electrons and energy obtained from
the oxidation of H2. Producing biodiesel-equivalent electrofuel
from renewable CO2 and H2 has immense potential, especially
if the fuel is compatible with the existing fuel infrastructure. This
research addressed enhanced substrate utilization by focusing on
two strategies: (1) optimizing transcriptional regulations to afford
over-expression of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the enzyme responsible for assimilation of CO2
into the CBB cycle; and (2) hydrogenase over-expression by introduction of additional copies of genes encoding a membrane-bound
hydrogenase (MBH), a soluble hydrogenase (SH), and their maturation machinery to enhance oxidation of H2 to generate NAD(P)
H and ATP required for CO2 fixation. Incorporation of these strategies into a single production strain resulted in 6-fold CO2 and
3-fold H2 uptake improvement, in vitro, with the overarching goal
of providing abundant reducing equivalents towards the economic
production of biodiesel in C. necator.
37
Sunday Afternoon
BIOT 53 – 4:00 p.m.
BIOT 60 – 3:40 p.m.
Engineering acetogenic Clostridia for n-butanol production
from CO2
Identification of a gene involved in the biosynthesis of
1-alkenes in the cyanobacterium Synechococcus sp. PCC7002
Chih-Chin Chen, [email protected], Shang-Tian Yang.Department of Chemical & Biomolecular Engineering, The Ohio State University,
Columbus, Ohio 43210, United States
Brian F Pfleger1,2, [email protected], Matthew B Begemann1,
Daniel Mendez-Perez1, [email protected]. (1) Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison,
Wisconsin 53706, United States (2) Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin 53706,
United States
Some Clostridia, including C. ljungdahlii and C. carboxidivorans,
are capable of producing ethanol and butanol from CO2 and H2.
However, the alcohol productivity, yield, and titer from these native strains are low and uneconomical for industrial applications. In
contrast, several acetogenic Clostridia can convert CO2 and H2 to
acetate with high yield, titer, and productivity comparable to those
from glucose as the substrate. A high metabolic flux from CO2 to
acetyl-CoA, the immediate precursor for both acetate and ethanol,
is the prerequisite for developing a highly productive ethanol and
butanol producers. In this work, we engineered homoacetogens for
ethanol and butanol production by introducing aldehyde/alcohol
dehydrogenase and genes in the acetyl-CoA to butyryl-CoA pathway using modular clostridia plasmids. The fermentation kinetics
of these engineered mutants and their ability to produce ethanol
and butanol from CO2 and H2 were studied and will be presented
in this paper.
BIOT 59 – 3:00 p.m.
Electrofuel production using chemolithoautotrophic iron
oxidizing bacteria in a reverse microbial fuel cell
Timothy M Kernan, [email protected], Asli Sahin, Bin Lin, Alan
C West, Scott A Banta, [email protected] of Chemical
Engineering, Columbia University, New York, NY 10027, United States
Biofuels made from electrochemical energy may have several practical advantages as compared to traditional photosynthetic pathways. The critical challenge in this approach is the electrochemical/
biological interface. We have been exploring the use of chemolithoautotrophic bacteria that are able to grow on easily reducible substrates. We have been developing a process using the ammoniaoxidizing bacteria Nitrosomonas europaea. Here we present a new
electrofuels platform using Acidothiobacillus ferrooxidans which
oxidizes ferrous iron to ferric. The iron is easily reduced electrochemically which allows for biomass production from electricity
and air. Initial findings demonstrate that A. ferrooxidans is a tractable microorganism with a robust metabolism doubling once every 14 hours. Growth rates in electrochemically reduced iron media are indistinguishable from rates in fresh media prepared. We
are currently engineering the cells to produce isobutanol, a drop-in
advanced biofuel. Reverse microbial fuel cells made with A. ferrooxidans will be a valuable platform for electrobiosynthesis.
38
Carbon dioxide emissions and the increasing demand for transportation fuels are encouraging the development of renewable alternatives to fossil fuels. Cyanobacterial hydrocarbons are a promising
alternative because the direct conversion of sunlight and carbon
dioxide to lipids will by-pass recalcitrant intermediates found in
terrestrial biomass. It has been reported that the cyanobacterium
Synechococcus sp. PCC7002, which is among the fastest growing
of all cyanobacteria, synthesizes two alkenes of unknown structure but nothing was known about their biosynthesis. In this study,
we characterized the lipid profile of Synechococcus sp. PCC7002
and demonstrate the involvement of a gene in the biosynthesis of
two C19 alkenes. The gene encodes a large multi-domain protein
with homology to Type I polyketide synthases, suggesting a new
route for hydrocarbon biosynthesis from fatty acids via an elongation-decarboxylation mechanism. The combinatorial nature of
polyketide synthesis raises the possibility of using this type of genes
to produce a wide range of industrially relevant 1-alkenes and high
value chemical building blocks.
BIOT 61 – 4:00 p.m.
Towards biofuel production in Synechocystis sp. PCC 6803:
Expanding the molecular biology toolbox
Stevan Albers2, Yi Ern Cheah1, Christie A.M. Peebles1,2, [email protected]. (1) Department of Chemical and Biological
Engineering, Colorado State University, Fort Collins, CO 80523, United
States (2) Graduate Program in Cell and Molecular Biology, Colorado State
University, Fort Collins, CO 80523, United States
Biofuel production in microorganisms has great potential to fulfill
this nation’s need for fungible “green” transportation fuels. Because
of the simplicity of bacterial cells, photosynthetic cyanobacteria
make good candidates as a platform for biofuel production. Synechocystis sp. PCC 6803 is an attractive production platform due
to rapid doubling times, a sequenced genome and an established
molecular biology toolbox. This molecular biology toolbox was established to probe the biology of photosynthesis and has not been
optimized for pathway engineering which is necessary for biofuels
production. This talk will discuss our efforts to develop these tools.
In particular, it will focus on our efforts to develop alternative
counter selection markers (to transform wild type Synechocystis)
and our efforts to develop and characterize promoters for gene expression under varying conditions.
BIOT 62 – 4:20 p.m.
Moving beyond traditional synthetic biology chassis:
Cyanobacteria as metabolic engineering platforms
Alex S Beliaev1, [email protected], Allan E Konopka1, Donald
A Bryant2, Jennifer L Reed3. (1) Biological Sciences Division, Pacific
Northwest National Laboratory, Richland, WA 99352, United States (2)
Department of Biochemistry & Molecular Biology, The Pennsylvania State
University, University Park, PA 16802, United States (3) Chemical &
Biological Engineering Department, University of Wisconsin, Madison, WI
53706, United States
Engineering biological systems for a variety of environmental and
industrial processes requires platforms with capabilities and complex adaptations that cannot be readily introduced into traditional
chassis (i.e., Escherichia coli, Saccharomyces cerevisiae). It obviates a need for cross-cutting approach that not only leverages new
genomic tools and design principles but also takes advantage of
systems biology methods to interrogate the genetic and functional
diversity at a whole-organism level. Using model cyanobacterial
systems (Synechococcus and Cyanothece spp.), we are conducting
systems-level analysis of modules involved in photosynthetic energy conservation and reductant generation; CO2 fixation; photosynthate production; and biosynthesis of metobolic intermediates
and monomers. Coupled with regulatory network reconstruction,
this approach is providing testable predictions of reductant fluxes,
reductant partitioning to carbon metabolism and other sinks as
well as anabolic and biosynthetic pathways that lead to macromolecular synthesis. This presentation summarizes the progress in
developing photoautotrophic platforms for metabolic engineering
and biotechnology applications.
BIOT 63 – 4:40 p.m.
Production of fungible diesel via fatty acid synthesis from
hydrogen and carbon dioxide feedstocks
Matthew L. Lipscomb1, [email protected], Tanya Warnecke
Lipscomb1, Hans Liao1, PinChing Maness2, Michael D. Lynch1. (1) OPXBIO, USA, United States (2) National Renewable Energy Lab, USA,
United States
There is increasing pressure to reduce dependence on foreign petroleum sources. As such, the development of green chemistry
routes to produce fuels from renewable feedstocks has been the focus of significant research. Traditional bio-refining processes rely
on microbial fermentation of renewable carbon sources such as
sugar into higher value products. More recently, work has focused
on the use of non-traditional feedstocks in bio-processing such as
cellulosic biomass, pyrolysis of waste biomass, or gasification of organic municipal solid waste, to name a few. OPXBIO is developing
a novel, engineered microorganism that produces free fatty acids of
targeted carbon chain length utilizing hydrogen (H2) and carbon
dioxide (CO2) as a feedstock. The free fatty acids are subsequently
converted to alkanes and or jet fuel using chemical catalysis. The
proposed process will fix CO2 utilizing H2 to generate an infrastructure-compatible, energy-dense fuel. The proposed process is
scalable, the initial economics are favorable, and the liquid fuel can
be used directly in the existing diesel infrastructure.
Stem Cells and Tissue Engineering:
Adult Stem Cells
2:00 p.m.
Room# 25A Harley, S. Palecek Papers 64-70
BIOT 64 – 2:00 p.m.
Cytoskeletal control of purity, proliferation,
and differentiation of human hematopoietic stem cells
Dennis E Discher, [email protected], Jae-Won Shin, Joe Swift,
Kyle Spinler, Amnon Buxboim.Chemical & Biomolecular Eng’g, University
of Pennsylvania, Philadelphia, PA 19104, United States
The cytoskeleton has essential roles in cell division, cotical stability, and firm adhesion to matrix, and so components such as
Non-muscle myosin-II (NMM-II) might be predicted to be low
in dormant hematopoietic stem cells (HSCs) and to increase with
differentiation. Deletion of NMM-II is known to be embryonic
lethal, but a role in HSC differentiation is uknown. Recently, we
showed that sustained pharmacological inhibition of NMM-II together with soft 2D matrices mimicking the perivascular niches in
marrow, rather than rigid like bone, maximizes both MK maturation and platelet generation in vitro and in vivo [Shin et al., PNAS,
2011; 108:11458-63]. HSCs exhibit some similarities to mature
MKs in that long-term HSCs do not divide in vivo while various
progenitors and maturing cells rapidly expand in number. Here,
reversible inhibition of NMM-II sustained over several cell cycles
enriches long-term HSCs up to 20 fold by selective elimination
of proliferating progenitors. Molecular profiling and functional
39
Sunday Afternoon
BIOT 58 – 2:40 p.m.
BIOT 65 – 2:20 p.m.
Regulatory machinery for combinatorial biological control
of erythroid progenitor cell fate decisions by stem cell factor
(SCF) and erythropoietin (EPO)
Julie Audet1,2,3, [email protected], Weijia Wang1,2,3, Vahe
Akbarian1,2,3. (1) University of Toronto, Canada (2) Institute of Biomaterials and Biomedical Engineering (IBBME), Canada (3) University of
Toronto, Donnelly Centre, Canada
Combinatorial cytokine control of stem/progenitor cells has an immense potential in regenerative medicine and for developing efficient stem/progenitor cell culture processes. However, due to the
heterogeneity and scarcity of these cell populations, little is known
about regulatory machinery underlying their combinatorial cytokine dependences. Erythrocyte colony-forming units (CFU-Es) are
SCF and EPO-responsive progenitor cells present in the adult bone
marrow and fetal liver. Since CFU-Es can be expanded and differentiated into red blood cells (RBCs), they are important potential
targets not only for the treatment of blood disorders, but also for the
manufacture of RBCs. In this presentation, I will describe how we
have examined the combinatorial control of CFU-E survival, proliferation and differentiation by SCF and EPO using i) multicolor
phospho-specific flow cytometry of single c-Kit+CD71highTer119cells and ii) carboxyfluorescein diacetate succinimidyl ester timeseries in association with a model of cell proliferation dynamics
which included generation-specific rate parameters.
40
BIOT 66 – 2:40 p.m.
Culture-associated mechanical changes of human
mesenchymal stem cells
Daniel McGrail1, [email protected], Kathleen McAndrews1,
Kevin Rodriguez1, Michelle R Dawson1,2, [email protected].
edu. (1) Department of Chemical and Biomolecular Engineering, Georgia
Institute of Technology, Atlanta, GA 30332, United States (2) Department
of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA
30332, United States
Of the many adult stem cells currently being investigated, mesenchymal stem cells (MSCs) show unique promise. They are easily
isolated and exhibit an innate immunosuppressive capacity making them ideal for transplantation. Furthermore, unlike many adult
stem cells, MSCs are easily expanded ex vivo to gather sufficient
numbers for transfusion. Due to recent studies demonstrating
the importance of cellular mechanical properties for a variety of
therapeutic properties ranging from migration to differentiation,
we sought to investigate if MSCs underwent mechanical alterations
during this ex vivo expansion. Typically, loss in therapeutic viability
can be detected by a variety of assays for cellular senescence. Here,
we show even before detection of these markers, MSCs undergo a
series of mechanical changes including alterations in morphology,
adhesion, contraction, intracellular rheology, and migration that
ultimately hinder in vivo therapeutic efficacy as demonstrated in a
xenographic wound healing model.
BIOT 67 – 3:00 p.m.
Biomolecular and structural cues in collagen-GAG scaffolds
alter mesenchymal stem cell bioactivity for tendon-bone interface repair
Brendan A Harley1,3, [email protected], Steven R Caliari1,
Daniel W Weisgerber1, Rebecca A Lyons2, Douglas O Kelkhoff1,3. (1) Department of Chemical and Biomolecular Engineering, University of Illinois
at Urbana-Champaign, Urbana, IL 61801, United States (2) Department
of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL
61801, United States (3) Instiute for Genomic Biology, University of Illinois
at Urbana-Champaign, Urbana, IL 61801, United States
Clinical approaches for repairing tendon injuries do not regenerate
the tendon-bone junction (TB). The native TBJ contains regionspecific compartments as well as gradients of matrix proteins and
soluble biomolecules across the interface. Based on this heterogeneity we hypothesized that overlapping patterns of tendon, bone,
and interface specific matrix proteins and soluble factors are required for regeneration of the anatomic insertion between tendon
and bone. We have developed a homologous series of collagenGAG (CG) biomaterials to assess the significance of microstructural as well as ECM protein and soluble biomolecule cues on
mesenchymal stem cell bioactivity. Scaffold anisotropy, pore size,
mineralization, and soluble biomolecule supplementation have
been shown to regulate mesenchymal stem cell lineage specification towards, and long-term maintenance of, distinct tendinous,
osseous, and interface phenotypes. Ongoing work is quantifying
the potential of coincident matrix and biomolecule cues to drive
regionally-distinct MSC differentiation and tissue biosynthesis in
a single CG scaffold.
BIOT 68 – 3:40 p.m.
Molecular engineering of synthetic stem cell niches
David Schaffer, [email protected] of Chemical and
Biomolecular Engineering, University of California, Berkeley, Berkeley, CA
94720-3220, United States
Stem cell microenvironments present complex repertoires of signals to regulate the processes self-renewal and differentiation.
There has been considerable progress in studying soluble signals
that regulate stem cell function, but comparatively less work has
focused on investigating the “solid phase” of the microenvironment, in large part due to experimental complexities in manipulating a complex mixture of proteins known at the extracelluar matrix
(ECM) and other components. We have used modular, bioactive
materials to investigate the role of matrix modulus or stiffness on
stem cell function, nanoscale organization of biochemical cues on
neural stem and human embryonic stem cell function, and the
combinatorial presentation of ECM ligands on cell behavior. Biomimetic materials can thus be employed to study mechanisms by
which the solid phase of a stem cell microenvironment regulates
cell function, as well as offer reproducible and scaleable systems to
control stem cells for biomedical application.
BIOT 69 – 4:20 p.m.
by an early upregulation of a key protein, the bone morphogenetic
protein-2 (BMP2), which is often supplemented in synthetic scaffolds to enhance bone formation. In this talk, we will present the
early effects of tobacco mosaic virus (TMV) on osteoinduction
with particular emphasis on its early BMP2 regulation.
BIOT 70 – 4:40 p.m.
Towards optimal culture conditions for the ex-vivo expansion
of umbilical cord blood hematopoietic stem/progenitor cells for
cell therapy
Pedro Z Andrade1, [email protected], Francisco dos
Santos1, Graça Almeida-Porada2, Cláudia Lobato da Silva1, Joaquim M.S.
Cabral1. (1) Department of Bioengineering, Institute for Biotechnology and
Bioengineering, Centre for Biological and Chemical Engineering, Instituto
Superior Técnico, Lisboa, Portugal (2) Wake Forest Institute of Regenerative Medicine, Winston Salem, North Carolina, United States
Ex-vivo expansion of hematopoietic stem/progenitor cells (HSC)
would be an enormous boost towards the widespread use of umbilical cord blood (UCB) for cell therapy applications, due to the
low HSC number available per UCB unit. In this study, UCB
CD34+-enriched cells were co-cultured with mesenchymal stem
cell (MSC)-derived feeder layers using a cytokine-supplemented
serum-free medium. Using a stem cell engineering approach, important parameters affecting ex-vivo expansion performance of
UCB HSC were systematically studied and optimized - serum-free
media formulations and cytokine cocktails, initial CD34+ cell enrichments - resulting in an increase of cell productivity (20-fold
increase in CD34+ cells after 7 days), while reducing culture costs
by 50-65% compared to previously established protocols. In addition, the effect of oxygen tension in HSC-MSC co-cultures was
investigated: physiological O2 levels (5-10%) were found to be beneficial for an efficient expansion of UCB HSC while cell metabolic
patterns were consistent with hypoxic adaptation, being shifted to
aerobic glycolysis.
Rapid osteoinduction by plant virus substrate mediated
through early upregulation of bone morphogenetic protein-2
Qian Wang, [email protected], Pongkwan Sitasuwan, L. Andrew
Lee, Sevan M Muhammad, Quyen L Nguyen, Elizabeth Balizan, Jittima
Luckanagul.Department of Chemistry and Biochemistry, University of
South Carolina, Columbia, SC 29208, United States
In recent years, how the nanoscale materials affect these complex
behaviors for applications in tissue engineering and regenerative
medicine are being vigorously investigated. One nanomaterial of
interest has been derived from plant viruses as cell supporting substrates, on which bone marrow derived mesenchymal stem cells
(BMSCs) were rapidly induced towards osteogenic cells. This enhanced differentiation on the virus coated substrates was preceded
41
Sunday Afternoon
analyses indicate that NMM-II isoforms play distinct roles during HSC differentiation. NMM-IIA is a marker for differentiation
with significantly lower expression in HSCs than committed progenitors, which is consistent with greater membrane flexibility of
HSCs measured by micropipette aspiration. In contrast, NMMIIB is 5 fold higher in HSCs and progenitors than differentiated
CD34- cells. HSC and progenitor numbers are also sensitive to matrix elasticity and density in a NMM-II dependent manner. Posttranslational modifications of NMM-IIA, specifically de-activating
and isoform-specific phosphorylation also occur differentially in
HSCs and progenitors, impacting cytoskeletal integrity,membrane
flexibility, and matrix sensing. Myosin-inhibited CD34+-derived
bone marrow cells maintain functional long-term HSCs in vivo in
the marrows of xenografted mice with an added benefit of increasing platelet circulation. Myosin-II inhibition and soft, high ligand
fibronectin constitute an important ‘microenvironment mimetic’
for enrichment of long-term HSCs, with Myosin-II being a central,
matrix-regulated node in hematopoiesis.
Proteins at Interfaces: Manufacturing, Formulation, and Delivery
2:00 p.m. Room# 25B
J. Champion, Y. Gokarn Papers 71-78
BIOT 71 – 2:00 p.m.
Coiled-coil leucine zipper peptides anchored onto lipid bilayers
for the design of temperature-sensitive liposomes
Zahraa Al-ahmady1, [email protected], Wafa’ Al-
Jamal1, Tam Bui2, Alex Drake2, James Mason2, Jeroen Bossche1, Kostas
Kostarelos1. (1) Nanomedicine Laboratory, Centre for Drug Delivery
Research, The School of Pharmacy, University of London, London, United
Kingdom (2) Biomolecular Spectroscopy Centre, King’s College London,
The Wolfson Wing, Hodgkin Building, Guy’s Campus, London,
United Kingdom
The present study describes a novel type of temperature-sensitive
liposome (TSL) system engineered by anchoring a coiled-coil leucine zipper peptide within the lipid bilayer to enhance drug release.
Circular Dichroism analysis showed that the peptide retained its
secondary structure and thermal-responsiveness after anchoring
into the lipid membrane with improved structural reversibility. In
addition, Lp-peptide vesicles showed a reduction in bilayer fluidity
at the inner-core as observed with DPH anisotropy studies, while
the opposite effect was observed with the ANS probe, indicating
peptide interactions with both regions of the membrane rather
than attaining only a superficial conformation. In vitro doxorubicin
release studied showed that the peptide anchoring into liposomes
improved their serum stability at physiological temperatures compared to equivalent molar concentration of cholesterol, without affecting the thermoresponsive nature of the system. In summary,
this study presents a promising new class of TSL which may open
new opportunities for mild hyperthermia triggered drug release.
42
BIOT 72 – 2:20 p.m.
Chiral vibrational sum frequency generation spectroscopy
allows real-time and in situ characterization of protein secondary structures at interfaces
Li Fu, Gang Ma, Elsa C. Y. Yan, [email protected] of
Chemistry, Yale University, New Haven, CT 06520, United States
Characterization of protein secondary structures at interfaces is
important to improve formulation, delivery, and manufacturing
processes of therapeutic proteins. Using surface-specific chiral
vibrational sum frequency generation (SFG) spectroscopy, we obtained amide I and N-H stretch spectra of various protein secondary structures at the air/water interfaces. The spectra show distinctive chiral vibrational signatures, which allow direct identification
of parallel beta-sheets, anti-parallel beta-sheets, alpha-helices, 3-10
helices, and random-coils. Because the spectra are muted to achiral
molecules at interfaces, the vibrational signatures are free of water
background. Using these signatures, we probed the misfolding of
an amyloid protein associated with type II diabetes at lipid/water
interfaces. We observed in real time and in situ that lipid induces
its conversion from random coil to alpha-helix and then betasheet. We conclude that chiral SFG can probe real-time kinetics of
protein conformational changes at interfaces, which will be useful
for developing treatments using therapeutic proteins.
BIOT 73 – 2:40 p.m.
Assessing the impact of thermal stability on protein adsorption
behavior using naturally occurring enzymes of the aldo-keto
reductase superfamily
Flora Felsovalyi1,2, [email protected], Tushar Patel1, Paolo
Mangiagalli3, Sanat Kumar1, Scott Banta1. (1) Chemical Engineering,
Columbia University, New York, New York 10027, United States (2) Pharmaceutical Systems, BD Medical, Franklin Lakes, New Jersey 07417, United
States (3) Pharmaceutical Systems, BD Medical, Pont de Claix, RhôneAlpes 38801, France
Elucidating mechanisms governing the behavior of proteins at
solid/liquid interfaces is particularly relevant in the interaction
of high-value biologics with storage and delivery device surfaces,
where adsorption-induced conformational changes may dramatically affect biocompatibility. The marked impact of structural stability on interfacial behavior has been investigated by engineering
non-wild-type stability mutants by artificially introducing disulfide
bonds. A potential shortcoming of such mutants is the inability to
decouple the mutation used to enhance stability from its effect on
adsorption. We employ two members of the aldo-keto reductase
superfamily to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its
subsequent impact on desorption. We report that thermostability
plays considerably different roles in the distinct kinetic legs of the
adsorption-desorption lifecycle. Although structural transitions
upon adsorption occur independent of thermostability, the latter
plays a major role in determining the reversibility pathway which
drives refolding upon desorption.
BIOT 74 – 3:00 p.m.
Impact of solution conditions on polysorbate oxidation and
mAb stability
Ankit R Patel, [email protected], Nina Xiao, Doris Lau, Mary
Krause, Jun Liu.Late Stage Pharmaceutical Development, Genentech, South
San Francisco, California 94080, United States
Surfactants such as polysorbate 20 are commonly used to protect
therapeutic proteins against stress occurring at the air-water interface. However, it is well known that the level required to protect
proteins from agitation-induced instability varies, and that polysorbates actually consist of a mixture of species and impurities
that have the potential to impact protein stability over time. In
this work, the effect of buffer species and polysorbate 20 level on
the stability of a monoclonal antibody (mAb) is examined under
thermally stressed conditions. In particular, it is shown that higher
levels of polysorbate can have deleterious effects on mAb and excipient stability under particular formulation conditions. These
results provide significant insight into the dominant degradation
mechanism of polysorbate at high temperatures and illustrate the
complex relationship between high polysorbate levels, buffer species, excipient oxidation, and protein aggregation.
BIOT 75 – 3:40 p.m.
Application of aqueous two phase systems to reduce protein
unfolding during microsphere fabrication and loading
Jerome V. Karpiak1, [email protected], Adah Almutairi1,2,4,3. (1)
Department of Biomedical Sciences, University of California at San Diego,
La Jolla, CA 92093-0600, United State (2) Skaggs School of Pharmacy and
Pharmaceutical Sciences, University of California at San Diego, La Jolla,
CA 92093-0600, United States (3) Department of NanoEngineering, University of California at San Diego, La Jolla, CA 92093-0600, United States
(4) Materials Science and Engineering Program, University of California at
San Diego, La Jolla, CA 92103-0600, United States
surface tension emulsion polymerizations. We are using these systems to fabricate pH- and light-responsive hydrogel microsphere
delivery vehicles loaded with active growth factors.
BIOT 76 - 4:00 p.m.
Effect of lectin-glycoprotein binding strength on glycoanalysis
Amit K Dutta1,2, [email protected], Alexander W Peterson1,
Huaying Zhao2, Hua-Jun He1, Rebecca Zangmeister1, Peter Schuck2, Michael J Tarlov1. (1) Biochemical Science Division, Material Measurements
Laboratory, National Institute of Standards and Technology, Gaithersburg,
MD, United States (2) NIBIB, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institutes of Health, Bethesda, MD,
United States
The mechanism of action of many monoclonal antibody (mAb)
therapeutics depends on the structure of the N–linked glycans
located on each heavy chain in the Fc region and, hence, precise
characterization of glycosylation is essential to ensure efficacy of
these mAb-based therapeutics. In this study, we used surface plasmon resonance (SPR) as a rapid, sensitive, and lower cost method
to assess the glycan structure of mAb therapeutics. Rituximab was
chosen as a model mAb; Concanavalin A (Con A), wheat germ
agglutinin (WGA) and peanut agglutinin (PNA) were chosen as
three model lectins. Rituximab binds with CD20 on B-cells and is
used to treat B-cell non-Hodgkin lymphomas resistant to chemotherapy. Con A and WGA bind with mannose (Ka= 0.25 µmol/L)
and N-acetyl glucosamine (GlcNAc, Ka= 2.5 mmol/L) residues,
respectively present in the glycan moiety of the mAb molecule.
PNA binds with Gal β-1,3-GalNAc residues, which are not present in rituximab, and was used as a control. Differences in binding
behavior between the model lectins and the glycans on the glycoprotein, rituximab, were found to be dependent on which protein,
the glycoprotein or the lectin, was covalently bound to the SPR
surface. We observed reversible binding behavior when a solution
of rituximab was introduced to surface bound Con A. In contrast,
irreversible binding behavior was observed when a solution of Con
A was introduced to surface bound rituximab. We also observed
that covalent immobilization of rituximab followed by injection
of WGA resulted in significant lectin-mAb binding, whereas immobilization of WGA followed by injection of rituximab did not
result in any significant binding. These results and others will be
presented along with theories to explain these observations.
Hydrogel microcarriers offer biocompatible solutions for protein
delivery. Typical methods for microsphere fabrication involve a
water-in-oil emulsion stabilized by surfactants. In such systems
interactions at the oil/water interface may affect tertiary protein
structure, and ultimately, bioactivity. We are optimizing aqueous
multiphase systems to allow partitioning of reactive macromers
and growth factors of interest into the discontinuous phase for low
43
Sunday Afternoon
Biophysical & Biomolecular
Processes:
BIOT 77 – 4:20 p.m.
Behavior of PEGylated proteins at oil/water interfaces relevant
to formulation in and delivery from poly(lactide-co-glycolide)
microspheres
Todd M. Przybycien, [email protected], Adam Canady, Robert
D. Tilton.Carnegie Mellon University, United States
While small molecule drugs realize delivery benefits, proteinbased drugs often fare poorly in poly(lactide-co-glycolide) (PLG)
microsphere depot systems due to interactions with the oil/water
(O/W) and solid/water (S/W) interfaces generated during formulation and release. Adsorption can be irreversible and can result in
protein denaturation, aggregation and, ultimately, bioactivity loss.
Protein PEGylation has been shown to mitigate this effect at the
S/W interface in addition to imbuing conjugates with several other
useful properties. We studied ribonuclease A (RNase A, a stable
protein) and apo-α-lactalbumin (ALA, a marginally stable protein)
grafted with 20kDa PEG chains at the ethyl acetate/water interface
to determine if PEGylation affords similar protection at typical
O/W interfaces. We used spectroscopic and optical techniques to
assess the adsorbed amount, structural state and aggregation state
of both unmodified and PEGylated proteins. ALA was found to
be more denatured than RNaseA, as expected. Surprisingly, PEGylation actually enhanced ALA denaturation.
BIOT 78 – 4:40 p.m.
Functional material based on mussel adhesive protein fused
with BC domain of protein A that efficiently immobilizes antibodies on diverse surfaces
Chang Sup Kim1, [email protected], Yoo Seong Choi2, Hyung
Joon Cha1. (1) Department of Chemical Engineering, Pohang University
of Science and Technology, Pohang, Kyungbuk 790-784, Republic of Korea
(2) Department of Chemical Engineering, Chungnam National University,
Daejeon, Republic of Korea
The efficient immobilization of antibodies onto solid surfaces is a
vital factor for the sensitivity and specificity of various immunoassays and immunosensors. In the present work, we designed and
produced a novel linker protein, BC-MAP, in Escherichia coli by
genetically fusing mussel adhesive protein (MAP) with two domains (B and C) of protein A (antibody binding protein) for efficient antibody immobilization on diverse surfaces. Through direct
surface coating analyses, we found that BC-MAP successfully coated diverse surfaces including glass, polymers, and metals, but the
BC domain alone did not. Importantly, antibodies were efficiently
immobilized on BC-MAP-coated surfaces, and the immobilized
antibodies interacted selectively with their corresponding antigen. Quartz crystal microbalance analyses showed that BC-MAP
44
has excellent antibody-binding ability compared to BC protein on
gold surfaces. These results demonstrate that the MAP domain,
with uniquely strong underwater adhesive properties, plays a role
in the direct and efficient coating of BC-MAP molecules onto diverse surfaces lacking any additional surface treatment, and the BC
domain of BC-MAP contributes to the selective and oriented immobilization of antibodies on BC-MAP-coated surfaces. Thus, our
BC-MAP fusion protein could be a valuable novel linker material
for the facile and efficient immobilization of antibodies onto diverse solid supports.
Monday Morning
Sessions
Monday Morning Sessions
A
B
C
D
E
8:30 a.m.
Room# 16A
Downstream Processes: Antibodies and Antibody-like Molecules
A.Ubiera, J. Neville Papers 79-86
8:30 a.m Room# 16B
Upstream Processes: Engineering Natural Products Biosynthesis
S. Garneau-Tsodikova, S. Ma Papers 87-94
8:30 a.m. Room# 17A
Advances in Biofuels Production: Applications of Systems Biology,
Synthetic Biology, and Metabolic Engineering
S. Atsumi, C. Trinh, H. Alper Papers 95-101
8:30 a.m.
Room# 25A Stem Cells and Tissue Engineering: Engineering of Stem Cell
Expansion and Differentiation
L. Lock, M. Dawson Papers 102-109
8:30 a.m. Room# 25B
Biophysical & Biomolecular Processes: Protein Characterization
Technologies – Structure, Stability, and Dynamics
J. Laurence, O. Stauch Papers 110-117
11:30 a.m.
Ali Khademhosseini
Room#16A
BIOT Young Investigator Award Lecture
45
Antibodies and Antibody-like Molecules
8:30 a.m.
Room# 16A
A.Ubiera, J. Neville Papers 79-86
parameters constant, and together with an industrial partner we
have successfully developed a new affinity resin ready for GMP use.
We anticipate that with this novel ALC resin as a capture step a
purification platform for lambda Fabs can be established.
BIOT 81 – 9:10 a.m.
Affinity purification of antibodies from biological fluids using
Fc-binding peptide ligands
BIOT 79 – 8:30 a.m.
Affinity platforms for the purification of antibody fragments
Carina Engstrand, [email protected], Björn Norén, Bengt
Westerlund.Chemistry, GE Healthcare, Uppsala, Uppsala 75184, Sweden
Monoclonal antibodies are typically purified with a platform approach where capture using affinity chromatography with Protein A has become the industry standard. However, for antibody
fragments there is not yet a corresponding solution. Here we will
discuss purification challenges for antibody fragments and will
present several chromatography media alternatives suitable for purification of antibody fragments of different subclasses, size, and
structure. Application examples showing the purification of antibody fragments using affinity media will be shown. Screening and
optimization of elution conditions, binding capacities, yield and
ligand leakage data will be presented, as well as challenges and opportunities of a platform purification solution for antibody fragments.
BIOT 80 – 8:50 a.m.
Building a purification platform for lambda Fabs: Experience
and strategy
Nora Eifler, [email protected] of BioProcess R&D,
Novartis Pharma AG, Basel, BS 4057 Basel, Switzerland
The biotechnology pipelines are of growing diversity. Antigenbinding fragments (Fabs) are novel formats in this pipeline. Sharing similar features to monoclonal antibodies (mAbs) with regards
to expression, Fabs are classified as ‘low risk’ for upstream development. Yet for downstream processing, the technologically mature
mAb platform is not directly applicable. New approaches therefore
need to be found in order to achieve a lean purification process
that maintains quality, productivity and timelines. To determine
concentration and purity, we have developed a cocktail of new
analytical methods tailored to meet our needs. Various resins were
screened for the capture step while keeping the other purification
46
Amith D. Naik2, [email protected], Stefano Menegatti2, Han-
nah R. Reese2, Ruben G. Carbonell1,2. (1) Biomanufacturing Training and
Education Center (BTEC), North Carolina State University, Raleigh, North
Carolina 27695, United States (2) Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina
27695, United States
Hexapeptide ligands HWRGWV, HYFKFD and HFRRHL identified by our group show high affinity for the Fc fragment of IgG. In
this work we demonstrate the applicability of these peptide-based
adsorbents to purify antibodies from cell culture supernatants,
transgenic plant extract and milk. The purity and yield of the MAbs
recovered from CHO supernatants were higher than 94% and 85%
respectively. The HWRGWV ligand was also used for purification
of MAb from plant extract. To prevent fouling of chromatographic
media by pigments and phenolic compounds, the plant extract was
pre-treated with a hydrophilically modified charcoal. This treatment removed pigments and phenolic compounds without significantly affecting the MAb concentration. The treated plant extract
was then subjected to affinity purification resulting in MAb purity
and yield of 96% and 90%. Finally, the peptide adsorbent was used
to purify antibody from milk and whey resulting in IgG purity and
yield of 95% and 85%.
BIOT 82 – 9:30 a.m.
Comparative characterization of monoclonal antibody
aggregates formed by two generation methods for use as
model feeds in downstream processing
William Cataldo, [email protected], Nazia Ahmed,
Christopher Gillespie, Mikhail Kozlov, Ajish Potty, Rong-Rong Zhu.Process
Solutions, EMD Millipore, Bedford, MA 01730, United States
Monoclonal antibody aggregates are the most significant product
related impurity and are often times immunogenic, requiring typical final formulation targets of <1%. Aggregates, especially dimers,
are inherently difficult to separate and pose significant challenges
in downstream purification demanding extensive method screening and process development. Part of the difficulty of developing
a dedicated and robust aggregate removal solution is the high
variability and often reversible nature of aggregates. In this work,
we develop an approach to generate a reliable, non-reversible aggregate-containing antibody feed stream that can be used for development of advanced purification solutions. Two independent
methods were developed: chemical cross-linking and pH shift. The
chemical cross-linking method was developed for generating covalent monoclonal antibody aggregates with a high ratio of dimer
to high molecular weight aggregates. The alternative method (pH
shift) developed to form non-reversible aggregates for comparison
used a hold at high pH and conductivity in order to simulate “process-induced aggregates.” The aggregates were characterized using
Protein A-HPLC, SDS-PAGE, SEC-HPLC, Weak Cation Exchange
Chromatography, Mass Spectrometry, and cIEF. To determine the
relevance of the induced aggregates from a process standpoint, the
model feeds and an inherent aggregate-containing feed were purified using an aggregate removal step. The similarities and differences in the performance of the aggregate removal step for the induced
and natural aggregate feeds will also be presented.
BIOT 83 – 10:10 a.m.
Impact of different elution modes on process robustness and
mass throughout
Suma Rao, [email protected], Gina Sperrazzo, Nick Keener, Oliver
Kaltenbrunner.Purification Process Development, Amgen, Thousand Oaks,
CA, United States
Over the past few years we have seen rapid advances in cell culture
capabilities with titers of 10 g/L being reported. Existing downstream facilities were designed for lower cell culture titers (1g/L)
and are now required to process much greater mass than originally
designed for. Downstream chromatography steps need to operate
at much higher loading to enhance mass throughput while providing appropriate separation. In this work we analyze the differences
between isocratic and gradient elution for ion-exchange columns
under high protein loading conditions. Particular attention is paid
to the effect produced by high protein loading on selectivity and
robustness and its implication on facility utilization, mass throughput and the ease of implementation in a manufacturing environment. How the choice of elution mode impacts manufacturing
constraints and capabilities will be discussed.
BIOT 84 – 10:30 a.m.
Adsorption and desorption of two mAbs were studied for two
cation exchangers: Nuvia S and UNOsphere S. UNOsphere S is a
macroporous matrix, and Nuvia S was obtained by incorporating
charged polymeric surface extenders on UNOsphere S backbone.
Side-by-side comparison showed larger binding capacities and
adsorption rates for Nuvia S. CLSM during adsorption showed
sharp intraparticle protein concentration profiles for UNOsphere
S, consistent with pore diffusion mechanism, but diffuse profiles
for Nuvia S, consistent with a diffusion mechanism driven by the
adsorbed protein concentration gradient. Batch and CLSM experiments shows lower desorption rates for Nuvia S. UNOsphere S
multicomponent adsorption pattern is consistent with pore diffusion and Nuvia S results are consistent with a model where protein
transport occurs through a hopping mechanism, in a single file diffusion fashion. These outcomes provides the knowledge to predict
the effects of operating conditions on adsorption, and desorption,
and, ultimately, on process performance.
BIOT 85 – 10:50 a.m.
Developing a preparative non-platform monoclonal antibody
purification to remove oxidation variants
Martha Lovato Tse1, [email protected], Shirin Fuller1, Lisa Gao1,
Angela Meier2. (1) Department of Purification Development, Genentech,
South San Francisco, California 94080, United States (2) Department of
Late Stage Cell Culture Development, Genentech, South San Francisco,
California 94080, United States
Upon assessment of a monoclonal antibody (MAb) for fit into Genentech’s purification platform a product variant was observed by
analytical size exclusion. The material in the peak was identified
as tryptophan oxidation variants present in harvested cell culture
fluid and throughout the purification process at levels greater than
5%. Hydrophobic interaction chromatography (HIC) was investigated to separate the oxidation variants from desired MAb. High
throughput screening of 12 HIC resins rapidly identified two resins for further evaluation. Process development on one HIC resin
afforded a step that decreased the oxidation variant to less than
1% with a step yield over 80%. The HIC step was combined with
optimized platform steps to produce an overall process capable of
delivering material meeting Phase I purity and quality targets. Development of this process afforded flexibility to the project and the
ability to produce the MAb with higher or lower levels of oxidation
variants.
Single component and multi-component behavior of mAbs on
cation exchangers with and without surface extenders
Ernie X Perez-Almodovar, [email protected], Yige Wu, Giorgio
Carta.Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia 22904, United States
47
Monday Morning
Downstream Processes:
Studies on the formation and purification of a monoclonal
antibody species containing a third light chain
Rachel B. Wollacott, [email protected], Trevor J. Morin, Glenn
Godwin, John Que, William Thomas, Sadettin S. Ozturk.MassBiologics,
Mattapan, MA 02126, United States
Size exclusion HPLC analysis of a human monoclonal antibody
showed the presence of a new MAb species (referred to as “shoulder”) that eluted with a retention time between the dimeric and
monomeric species of the antibody. Extensive characterization of
this species using analytical methods indicated that it was a MAb
containing an extra light chain (molecular weight ~175 kDa). Interestingly, the amount of shoulder (typically 1-3% of the total MAb
present) varies with the CHO cell line producing the MAb and to a
lesser extent shoulder abundance is also influenced by the growth
conditions. Ion exchange chromatography (AEX, CEX) or hydrophobic interaction chromatography (HIC) are commonly used as
polishing steps to remove contaminants from monomeric MAb
preparations. Attempts to purify the high molecular weight species
from our MAb using our AEX → CEX platform was successful at
removing dimer and higher molecular weight species, but not sufficient for removing the shoulder contaminant. It was determined
that HIC could be used in place of CEX to exploit the subtle differences in hydrophobicity between monomer and shoulder. We have
developed an antibody polishing process on Butyl Sepharose HP
resin capable of removing the majority of high and low molecular
weight contaminants yielding 99% pure MAb monomer with a step
recovery of about 80%.
Upstream Processes:
Engineering Natural Products Biosynthesis
8:30 a.m Room# 16B
S. Garneau-Tsodikova, S. Ma Papers 87-94
BIOT 87 – 8:30 a.m.
Biosynthesis of highly modified UMP-derived nucleosides,
potential new antibiotic scaffolds
Steven G. Van Lanen1, [email protected], Xiuling Chi2, Koichi
Nonaka3. (1) Department of Pharmacy, University of Kentucky, United
States (2) Department of Life Sciences, University of Michigan, United
States (3) Biopharmaceutical Research Group I, Daiichi Sankyo, Co.
Ltd., Japan
48
Several nucleoside antibiotics have been discovered the past decade by using an activity-based screen to identify inhibitors of
bacterial translocase I, an essential enzyme involved in the biosynthesis of peptidoglycan cell walls. These nucleosides all consist
of a highly modified uridine with unusual sugar and, in several
instances, peptide appendages. One series of these nucleosides,
which includes caprazamycins, muraymycins, and liposidomycins,
are O-glycosylated with 5-amino-5-deoxyribose at C-5’ of the core
nucleoside to form a diriboside scaffold. We have now delineated
the biosynthetic mechanism for the 5-amino-5-deoxyribose using
the biosynthetic genes involved in the assembly of A-90289, a liposidomycin analogue isolated from Streptomyces sp. SANK 60405.
This biosynthetic pathway features sequential catalysis by five enzymes: a non-heme, Fe(II)-dependent-ketoglutarate:UMP dioxygenase (LipL), a L-methionine:uridine-5’-aldehyde aminotransferase (LipO), a 5’-amino-5’-deoxyuridine phosphorylase (LipP), an
UTP:5-amino-5-deoxy-D-ribose-1-phosphate uridylyltransferase
(LipM), and a 5-amino-5-deoxyribosyltransferase (LipN). This
pathway is highlighted by, among other features, a ribose unit that
is derived via initial 5’-oxidation of uridine-5’-monophosphate.
Mechanistic studies on the enzymes from this and related sugar
biosynthetic pathways will be presented.
BIOT 88 – 8:50 a.m.
Meta-omic characterization of the marine invertebrate
microbial consortium that produces the chemotherapeutic
natural product ET-743
Christopher M. Rath1, [email protected], Benjamin Janto2, Josh
Earl2, Azad Ahmed2, Fen Z. Hu7, Luisa Hiller2, Meg Dahlgren7, Rachel
Kreft2, Fengan Yu1, Jeremy J. Wolff5, Hye Kyong Kweon8, Michael A.
Christiansen6, Kristina Hakanasson8, Robert M. Williams6, Garth D. Ehrlich2,3, David H. Sherman1. (1) Department of Life Sciences, University
of Michigan, United States (2) Center for Genomic Sciences, AlleghenySinger Research Institute, Allegheny General Hospital, United States (3)
Department of Microbiology and Immunology, Drexel University College
of Medicine, Drexel University College, United States (4) Department of
Otalaryngology, Head and Neck Surgery, Drexel University College of Medicine, Drexel University College of Medicine, United States (5) Department
of Biological Chemistry, Bruker Daltonik, United States (6) Department of
Chemistry, Colorado State University, United States (7) Allegheny-Singer
Research Institute, Allegheny General Hospital, United States (8) Department of Biological Chemistry, University of Michigan, United States
In many macroorganisms, access to biologically active natural
products has not been possible due to an inability to identify and
culture producing microorganisms. We developed a meta-omic approach to identify and characterize the ET-743 (Yondelis®) biosynthetic pathway. This approved anti-cancer drug is obtained from
the tunicate Ecteinascidia turbinata, and clinically supplied by a
lengthy semi-synthetic process. Using metagenomic sequencing of
total DNA from the tunicate/microbial consortium we assembled
the core biosynthetic pathway from the producing bacteria Candidatus Endoecteinascidia frumentensis. Metaproteomics analysis
confirmed expression of biosynthetic proteins and the activity of
a key enzyme involved in assembly of the tetrahydroisoquinoline
core of ET-743 was verified in vitro. This work provides a foundation for direct production of the drug and new analogs through
metabolic engineering in an amenable host. We expect that this
interdisciplinary approach is applicable to diverse host-symbiont
systems that generate valuable natural products for drug discovery
and development.
quired flexibility and access. Overexpression of this FAS allowed
growth of a yeast strain deficient in fatty acid synthesis; thereby,
demonstrating in vivo activity and full functional replacement of
the native FAS. By removing the thioesterase domain and overexpressing novel short chain thioesterases, this enzyme enabled the
production of short chain fatty acids in yeast.
BIOT 89 – 9:10 a.m.
Gavin J Williams, [email protected], Irina Koryakina, Zhixia
Ye, John McArthur.Department of Chemistry, North Carolina State University, Raleigh, NC 27695-8204, United States
Burkholderia emerging as the next treasure island of
natural products
Yi-Qiang (Eric) Cheng, [email protected] of Chemistry
and Biochemistry, University of Wisconsin, United States
Burkholderia are Betaproteobacteria that are often associated
with human or animal diseases called “melioidosis”. The genomes
of more than 50 Burkholderia species or isolates have been sequenced, which revealed not only many anticipated pathogenic
determinants but also a lot of unexpected secondary biosynthetic
genes and gene clusters. Recent efforts from researchers in the biochemical field have discovered more than two dozens of new natural products (small molecules) from a few of the Burkholderia species or isolates; some of those molecules have interesting structures
and promising antibiotic or anticancer activities. This presentation
will provide a summary count of recent discoveries of new natural
products from Burkholderia by others and by our own research. It
appears that Burkholderia could become the next treasure island
of natural products.
BIOT 90 – 9:30 a.m.
Development of Saccharomyces cerevisiae strains for the
synthesis of short chain fatty acids
Christopher T Leber, [email protected], Nancy A Da Silva.Chemical
Engineering and Materials Science, University of California, Irvine, Irvine,
CA 92697, United States
Saccharomyces cerevisiae strains were engineered to produce
short chain fatty acids. These saturated carboxylic acids can be
transformed through chemical catalysis to intermediates currently
derived from petroleum based feedstocks. The synthesis of short
chain fatty acids requires access of short chain thioesterases to the
growing fatty acid chain. The complex and closed structure of the
native yeast fatty acid synthase (FAS) prevents this. Therefore, we
have introduced a heterologous type-1 FAS that provides the re-
BIOT 91 – 10:10 a.m.
Reprogramming the biosynthesis of natural products by directed evolution
Directed evolution is a powerful strategy for altering the
function and properties of enzymes. We describe our combined
efforts aimed at improving the utility of combinatorial biosynthesis
by reprogramming the biosynthesis of natural products by evolutionary strategies. Areas of focus include 1) improving the promiscuity of tailoring enzymes which modify natural product scaffolds,
2) improving the modularity of biosynthetic machinery, and 3)
developing new screening methods for evolution of enzymes involved in natural product biosynthesis.
BIOT 92 – 10:30 a.m.
Precursor-centric genome mining approach to novel lasso
peptide discovery
Mikhail Maksimov, [email protected], A. Jamie Link.Department of Chemical and Biological Engineering, Princeton University, United
States
Lasso peptides are a class of ribosomal natural products (RNPs)
that have a unique fold, exceptional stability and a wide range of
function. Their biosynthesis relies on the action of two enzymes
that mature the precursor peptide into its final fold. Implementation of homology based searches for discovering novel lasso peptides is problematic due to the short length of the precursor, as
well as, the lack of consensus sequences for the maturation machinery. Furthermore, experimental approaches aimed at isolating
novel lasso peptides from known RNP producers is difficult due
to potential gene silencing in the host organism. Fortunately, the
structural features of lasso peptides restrict the sequence space of
the precursor in a way that can be exploited to identify new lasso
peptide gene clusters. We will present the results of this precursorcentric approach to identify novel lasso peptides in sequenced bacterial genomes. In addition, we will describe our experimental efforts toward heterologous expression of these novel lasso peptides
in E. coli.
49
Monday Morning
BIOT 86 – 11:10 a.m.
Engineering the adenylation domain of DhbE to expand its
substrate specificty for entrobactin biosynthesis
Jun Yin, [email protected] of Chemistry, University of
Chicago, Chicago, IL 60637, United States
Engineering the substrate specificity of the “gate-keeping” enzymes in the biosynthetic pathways could enable the incorporation of nonnative building blocks into the natural product scaffold.
We recently developed phage display and yeast cell surface display
methods to engineer the substrate specificity of the adenylation (A)
domain of nonribosomal peptide synthetase (NRPS). In this talk I
will report our progress on engineering the A domain of DhbE to
expand its substrate specificity for enterobactin biosynthesis.
BIOT 94 – 11:10 a.m.
Genome mining of polyketides and nonribosomal peptides
in Aspergillus
Clay C. C. Wang, [email protected] of Pharmacy, University
of Southern California, Los Angeles, CA 90089, United States
Aspergillus species are known to produce high value secondary
metabolites such as lovastain. Recently the genomes of several Aspergillus species have been sequenced. One of the most important
findings from these genome efforts is the realization that these
organisms have the potential to produce far more secondary metabolites than have ever been isolated and identified. I will present
our recent efforts in identifying new secondary metabolites and
their corresponding biosynthesis pathways from several different
Aspergillus species. Finally I will present approaches to use natural
products we have isolated from Aspergillus as starting point for
further drug discovery.
Advances in Biofuels Production:
Applications of Systems Biology, Synthetic
Biology, and Metabolic Engineering
8:30 a.m. Room# 17A
S. Atsumi, C. Trinh, H. Alper Papers 95-101
BIOT 95 – 8:30 a.m.
Engineering fatty acid biosynthesis for the production of
sustainable fuels and chemicals
Bernardo M. T. da Costa, [email protected], Inc., South San Francisco, CA 94080, United States
It is widely recognized that sustainable replacements for fossil fuels and chemicals are urgently needed. By harnessing the efficiency
of fatty acid biosynthesis and employing an engineering strategy
that places all chemical unit operations within a single whole cell
catalyst and allows easy product recovery, LS9 has enabled efficient
biosynthetic routes and processes to a diversity of sustainable fuels and chemicals, including esters, alcohols, alkenes, and alkanes.
LS9 uses state-of-the-art synthetic biology techniques to rapidly
design, construct and evaluate its engineered microbes in all LS9
programs. This presentation will focus on the design, construction,
and improvement of our microbial biocatalysts and progress in the
development and scale up of our fuel and chemical processes.
BIOT 96 – 8:50 a.m.
Using OptForce to customize metabolic interventions for the
overproduction of fatty acids C6 through C16 in
Escherichia coli
Sridhar Ranganathan1,4, [email protected], Ting Wei Wang2,4, Jesse
Welsh2,4, Jong Moon Yoon2,4, Jacqueline V. Shanks2,4, Ka-Yiu San3,4,
Costas D. Maranas4,5. (1) Huck Institutes of Life Sciences, The Pennsylvania State University, University Park, PA 16802, United States (2) Department of Chemical and Biological Engineering, Iowa State University, Ames,
IA 50011, United States (3) Department of Bioengineering, Rice University,
Houston, TX 77005, United States (4) Center for BioRenewable Chemicals
(CBiRC), Ames, IA 50011, United States (5) Department of Chemical
Engineering, The Pennsylvania State University, University Park, PA 16802,
United States
The native metabolic capability and amenability for genetic manip-
50
ulations make Escherichia coli highly suited for the biosynthesis of
fatty acids. In this talk, we present results from an integrated experimental and computational study aimed at improving the yield
of fatty acids in E. coli. Upon characterizing the flux distribution of
the wild-type strain, we deployed a customized version of the OptForce procedure to identify all fluxes that must change to ensure the
overproduction of fatty acids of varying chain lengths (C6 through
C16). Subsequently, we identify a minimal set of these reactions
that must be actively engineered (up-/down-regulated, or knocked
out) in order to guarantee a pre-specified yield for the fatty acids.
Strategies suggested by OptForce suggest chain-dependent interventions that not only maximize fatty acid precursors (e.g. acetylCoA, malonyl-ACP etc.) but also disruption of chain elongation to
prevent the production of longer-chain acids. Based on interventions suggested by OptForce, we have developed a strain of E. coli
that exhibits fatty acid yields close 80% of theoretical maximum.
BIOT 97 – 9:10 a.m.
Functional genomics guided strain improvement for fatty acid
overproduction in Escherichia coli
Brian F Pfleger1,2, [email protected], Rebecca M Lennen1,2, [email protected], Max A Kruziki1, Kritika Kumar1, Robert A
Zinkel2,3, Kristin E Burnum2,4, Mary S Lipton2,4, Spencer W Hoover2,
Don R Ranatunga2, Tyler M Wittkopp2, Wesley D Marner2. (1) Department of Chemical and Biological Engineering, University of WisconsinMadison, Madison, WI 53706, United States (2) U.S. Department of
Energy Great Lakes Bioenergy Research Center, Madison, WI 53706,
United States (3) University of Wisconsin Biotechnology Center, Madison,
WI 53706, United States (4) Biological Sciences Division, Pacific Northwest
National Laboratory, Richland, WA 99353, United States
Microbial overproduction of free fatty acids is one route to next
generation biofuels such as alkanes, olefins, and fatty acid esters.
Moderate titers of free fatty acids can be achieved by expression of
an acyl-acyl carrier protein thioesterase in E. coli, however a nextgeneration strain has not yet been engineered that can achieve
greater than 30% of the maximum theoretical yield. It was found
that fatty acid overproduction results in large reductions in cell viability, compromised membrane integrity, and changes in cell morphology. To determine a functional basis for these observations, a
differential transcriptomic, proteomic, and lipidomic analysis was
conducted that compared fatty acid overproducing strains to nonoverproducing control strains. Altered expression levels were evident for numerous genes and proteins related to membrane stresses, proton motive force dissipation, and cell envelope composition.
These findings and successive iterations of strain engineering based
on functional genomics guided hypotheses will be discussed.
BIOT 98 – 9:30 a.m.
13 C Metabolic flux analysis of Saccharomyces cerevisiae
under octanoic acid inhibition
Ting Wei Tee, [email protected], Jong Moon Yoon, Laura Jarboe, Jacqueline V. Shanks.Center of Biorenewables Chemical, Iowa State University,
Ames, Iowa 50011, United States
Short-chained fatty acids synthesized via fermentation from biorenewable feedstocks are a potential source of platform chemicals,
and thus could help replace the traditional petrochemical’s dependence on crude oil. However, toxicity of fatty acids is an obstacle
in the high titer of fatty acid production and it remains a key challenge in metabolic engineering. Metabolic flux analysis (MFA),
the quantification of fluxes in metabolic pathways, is an integral
tool for the development of strategies for genetic modification and
the identification of metabolic regulation, by comparing fluxes
under different environments. We used Saccharomyces cerevisiae
as a model system to study the effect of toxicity of octanoic acid.
The exposure of octanoic acid to yeast caused significant growth
inhibition. We elucidated the metabolic flux differences in central metabolism between control and octanoic acid inhibition by
conducting 13C labeling experiments using fermentors. The yeast
cultures were fed with a mixture of uniformly 13C labeled glucose
and 1-13C positional labeled glucose. We quantified glucose uptake rate and fermentation product secretion rate using HPLC. The
amino acid isotopomer fractions were measured using 2D [13C,
2H] HSQC NMR. Flux distributions were computed from simulating isotopomer distribution and then fitting it to the experimental
measurements. We found distinctions in central metabolism flux
distribution between control and treatment, especially in the TCA
cycle. These observations could be coupled with transcriptome
data to pinpoint the system bottleneck and identify the important
genes responsible to enhance fatty acid tolerance. This project was
funded by U.S. National Science Foundation (EEC-0813570).
BIOT 99 – 10:10 a.m.
Production of not-so-corny biofuels
James C. Liao, [email protected] of Chemical and Biological Engineering, University of California, Los Angeles, United States
Global energy and climate problems have stimulated increasing efforts in synthesizing fuels and chemicals from renewable resources.
Current biofuel research and development efforts focus on converting cellulosic materials to ethanol or algal lipids to biodiesels,
which have a number of issues and limitations. A heretofore unexplored solution to these limitations would be to use proteins as
a feedstock. We first developed a set of pathways that convert keto
51
Monday Morning
BIOT 93 – 10:50 a.m.
BIOT 100 – 10:50 a.m.
Microbial production of terpene-based advanced biofuels
Taek Soon Lee, [email protected] Synthesis Division, Joint BioEnergy
Institute, Emeryville, CA 94608, United StatesPhysical Bioscience Division,
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, United States
Isoprenoids are naturally occurring hydrocarbons with a branched,
and in many cases, a cyclic structure. They are produced mostly by
plant and have been used traditionally as medicine and fragrance
ingredients. However, recently the use of these molecules as biofuels has been explored and has shown a great possibility especially
with their great cold weather property to be used as Diesel or Jet
fuel alternatives. To use isoprenoids as biofuels, the production in
plants, the natural producer, may not be adequate due to the limitation of scalability and the land use issue. So the engineering of
microorganisms that can produce a large amount of isoprenoids
became an attractive option to the researchers in the biofuel field
recently.
In this report, we first designed and identified isoprenoid compounds, specifically monoterpenes (C10) and sesquiterpenes
(C15) that can be potentially used as a fuel. We have tested the appropriate fuel properties of the target compounds, and engineered
the heterologous biosynthetic pathway into two model hosts, E.
coli and S. cerevisiae, to produce these terpene compounds with
a relatively high yield. To achieve higher production titer of these
potential terpene fuel molecules, we have optimized the pathway
to accumulate the precursors using synthetic biology tools, and
also engineered the host strain to make fermentation process
more efficient.
BIOT 101 – 11:10 a.m.
Application of directed evolution of xylose transport and
catabolism in yeast
Hal S. Alper, [email protected], Eric Young, Sun-mi Lee.Department of Chemical Engineering, The University of Texas at Austin, Austin,
TX 78712, United States
52
Metabolic engineering and directed evolution are powerful approaches for controlling and improving the production of biomolecules from biomass sugars, especially xylose. Here we demonstrate
the application of directed evolution to two key steps in biomass
utilization: molecular sugar transporters and catabolic enzymes.
Specifically, metabolic flux is limited at the transport level by low
pentose affinity and hexose inhibition. Here, we present evidence
that heterologous transporters may be engineered using directed
evolution to improve S. cerevisiae pentose growth characteristics
and ultimately biofuels production. Likewise, xylose catabolic enzymes (including xylose isomerase) do not have sufficient activity
to support high level, redox-balanced xylose utilization. Here, we
present evidence that these enzymes can be evolved for improved
function in yeast. By employing directed evolution in both of these
cases, significant improvements in xylose growth rate and ethanol
production are achieved.
gated. In this study, we found that tumor-secreted soluble factors,
including transforming growth factor- β1 (TGF-β1) and plateletderived growth factor (PDGF), dramatically alter the morphology,
intracellular rheology, cytoskeletal organization, and adhesivity of
MSCs. These mechanical changes were correlated with increased
migration.
BIOT 103 – 8:50 a.m.
Mechanical derivation of functional myotubes from
adipose-derived stem cells
Yu Suk Choi1, [email protected], Ludovic G Vincent1, Andrew R Lee1,
Marek K Dobke2, Adam J Engler1. (1) Department of Bioengineering, University of California, San Diego, La Jolla, California 92093, United States
(2) Department of Plastic Surgery, University of California, San Diego, La
Jolla, California 92093, United States
Stem Cells and Tissue Engineering:
Engineering of Stem Cell Expansion and
Differentiation
8:30 a.m.
Room# 25A L. Lock, M. Dawson Papers 102-109
BIOT 102 – 8:30 a.m.
Soluble growth factor induced mechanical changes regulate the
migration of mesenchymal stem cells to tumors
Deepraj Ghosh, [email protected], Daniel McGrail, Kathleen
McAndrews, Michelle R. Dawson, [email protected]
of Chemical and Biomolecular engineering, Georgia Institute of Technology,
Atlanta, Georgia 30332, United States
Mesenchymal stem cells (MSCs) are excellent candidates for gene
delivery since they can be isolated from the bone marrow, expanded rapidly, genetically engineered for stable production of therapeutic proteins, and reinfused as gene delivery vectors. Natively,
MSCs migrate toward inflammatory mediators released by tumors;
however, ex vivo expansion and genetic manipulation reduce their
homing capacity. The identification of growth factors that stimulate their spontaneous incorporation in tumors may be a key element in the development of MSC-based therapeutics that can
overcome transport issues. Numerous studies have focused on the
molecular response of MSCs to soluble factors, but the mechanical
properties that govern their migration are yet to be fully investi-
Though reduced serum or myoblast co-culture alone can differentiate adipose-derived stem cells (ASCs) into mesenchymal lineages, efficiency is usually not sufficient to restore function in vivo.
Often when injected into fibrotic muscle, their differentiation may
be misdirected by the now stiffened tissue. Here ASCs are shown
to not just simply reflect the qualitative stiffness sensitivity of bonemarrow-derived stem cells (BMSCs) but to exceed BMSC myogenic capacity, expressing the appropriate temporal sequence of muscle transcriptional regulators on muscle-mimicking extracellular
matrix in a tension and focal adhesion-dependent manner. ASCs
formed multi-nucleated myotubes with a continuous cytoskeleton
that was not due to misdirected cell division; microtubule depolymerization severed myotubes, but after washout, ASCs re-fused at
a rate similar to pretreated values. BMSCs never underwent stiffness-mediated fusion. ASC-derived myotubes, when replated onto
non-permissive stiff matrix, maintain their fused state. Together
these data imply enhanced mechanosensitivity for ASCs, making
them a better therapeutic cell source for fibrotic muscle.
BIOT 104 – 9:10 a.m.
Synthetic bone grafts as an instructive microenvironment for
osteogenic differentiation of adult stem cells
Ameya Phadke1, [email protected], Yuru Shih1,2,3, Koi-
chi Masuda4, Shyni Varghese1. (1) Bioengineering, University of California- San Diego, La Jolla, CA 92093, United States (2) Stem Cell Research
Center, National Yang-Ming University, Taipei, Taiwan Republic of China
(3) Institute of Clinical Medicine, National Yang-Ming University, Taipei,
Taiwan Republic of China (4) Department of Orthopedic Surgery, University of California- San Diego, La Jolla, California 92093, United States
use of autologous bone and/or expensive recombinant growth
factors. We have developed bone-mimicking composite materials, consisting of three-dimensional hydrogels mineralized with
an apatite-like mineral phase through a templating process. These
scaffolds were found to promote the attachment, proliferation and
osteogenic differentiation of human mesenchymal stem cells (hMSCs) even in the absence of osteogenic supplements. Specifically,
we determined that these mineralized matrices likely promoted
differentiation of hMSCs through a combination of physicochemical cues. Upon subcutaneous implantation in nude rats, the mineralized scaffolds were found to promote neovascularization into the
scaffold interior, accompanied by formation of bone tissue within
9 weeks. These osteoinductive grafts could be a cost-effective alternative to the use of expensive recombinant growth factors for bone
grafting.
BIOT 105 – 9:30 a.m.
Modeling and analysis of the core architecture regulating
TGFß induced epithelial to mesenchymal transition (EMT)
Anirikh Chakrabarti1, [email protected], Russell Gould2,
Jonathan T Butcher2, Jeffrey D Varner1. (1) Chemical and Biomolecular
Engineering, Cornell University, Ithaca, NY 14853, United States (2) Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853,
United States
Epithelial to mesenchymal transition (EMT) is important in development and pathological processes such as fibrosis and cancer.
Several extracellular signals trigger EMT, e.g., soluble transforming
growth factor ß (TGFß) family members. Receptor mediated signaling in response to TGFß triggers a program that ultimately represses the expression of epithelial genes such as E-cadherin, while
simultaneously activating the expression of mesenchymal genes
such as Vimentin. In this study, we developed a dynamic network
model of TGFß induced EMT signaling. Our model used mass
action kinetics within an ordinary differential equation (ODE)
framework to describe the EMT signaling and gene expression
program initiated by TGFß isoforms. The interaction network contained 995 protein or mRNA components interconnected through
1700 interactions. Signal flow, sensitivity, and robustness analysis,
suggested three regulatory nodes critical to TGFß induced EMT
as AP1/SP1, MAPK and LEF1. Alterations/malfunctions of these
nodes revealed possible operational paradigms of phenotypic conversion.
Synthetic bone-mimetic materials represent a promising off-theshelf alternative to current bone grafting strategies, which involve
53
Monday Morning
acids to higher alcohols. We further apply metabolic engineering to
generate Escherichia coli that can deaminate protein hydrolysates,
enabling the cells to convert proteins to higher alcohols. We accomplish this by introducing three exogenous transamination and
deamination cycles, which provide an irreversible metabolic force
that drives deamination reactions to completion. These results
show the feasibility of using proteins for biorefineries, for which
high-protein microalgae could be used as a feedstock with a possibility of maximizing algal growth and total CO2 fixation.
Microarray amplification of natural directional persistence
Girish Kumar, Carlos Co, Chia-chi Ho, [email protected] of Chemical Engineering, University of Cincinnati, Cincinnati, OH
45221, United States
Cell locomotion plays key roles in embryonic morphogenesis,
wound healing, and cancer metastasis. Here we show that intermittent control of cell shape using microarrays can be used to amplify
the natural directional persistence of cells and guide their continuous migration along preset paths and directions. The cell polarity can be induced by a range of shapes and the Golgi orientation
dynamics were characterized. The cell polarization, induced by the
asymmetric shape of individual microarray islands, is retained as
cells traverse between islands. Vary the size of the adhesive island
changes the tendency of cell migration. The study provide insights
on the role of cell morphology in directional movement and the
design of micropatterned materials for steering cellular traffic.
BIOT 107 – 10:30 a.m.
Differentiation and biomineralization of dental pulp stem cells
(DPSCs) on the cross-linked gelatin hydrogels for bone
regeneration
Divya Bhatnagar1, [email protected], Aneel Bherwani2, Miriam Rafailovich1, Marcia Simon2. (1) Department of Materials
Science and Engineering, Stony Brook University, Stony Brook, New York
11794, United States (2) Department of Oral Biology and Pathology, Stony
Brook University, Stony Brook, New York 11794, United States
This work investigates the differentiation in the absence of chemical induction, mechanical stimuli and only resulting from the stimuli of the substrate chemistry. We chose enzymatically cross-linked
gelatin hydrogels substrates of different stiffness varying from 8KPa
to 100Pa. DPSCs were cultured on the substrates for 7, 14 and 21
days with and without dexamethasone induction media. SEM and
EDX analysis after 21 days indicate that cells produced a sheet of
biomineralized deposits, several tenths of mm thick irrespective of
substrate stiffness and chemical induction. Modulli of the cells was
also independent of the induction and stiffness of the hydrogels.
RT-PCR assays indicated that cells expressed more osteocalcin
when cultured in non-induction media and harder substrate. Further experiments indicated that conformational change due to the
crosslinking of gelatin could be the reason for biomineralization.
BIOT 108 – 10:50 a.m.
Human embryonic stem cell derivates for clinical application
Hans S. Keirstead, [email protected] and
Neurobiology, University of California, Irvine, Irvine, CA 92697,
United States
California Stem Cell has generated a clinical grade product
of motor neuron progenitors derived from hESCs suitable for
addressing lower motor neuron loss. Populations typically consist
of greater than 95% young motor neurons; definitive morphologic
markers include Isl1, Hb9, NeuN, Tuj1, and SMI32, and functional
assessments including electrophysiology and functional membrane
channel responses. The final product is free from pluripotent,
undifferentiated cells, does not elicit an immune response, and
is quality controlled for sterility and karyotypic stability. The
clinically compliant differentiation method involves a chemically
defined media formulation free from animal components, avoids
the use of multiple and non-physiological doses of growth factors,
and avoids extensive in vitro expansion and dissociation cycles.
Manufacture is conducted under current good manufacturing
practices, including quality controlled storage and characterization
of the originating embryonic stem cells, procurement of reagents,
media manufacture, propagation, in-process control points and
batch release criteria of the final product.
BIOT 109 – 11:10 a.m.
High throughput screening of survivin expression modulators
associated with cytotoxic and embryotoxic effects
Ru Zang, [email protected], shang-Tian Yang.department of chemical &
biomolecular engineering, The Ohio state university, columbus, OH 43210,
United States
Survivin belongs to IAP family and is over-expressed in embryonic
stem cells and malignant cancer cells. Several studies found that
survivin was biologically important for oocyte development and
54
maturation. In this work, we developed an in vitro three-dimensional
fluorescent embryonic stem cell-based model for indentifying
potential survivin modulators in high throughput manners. We
hypothesized that decreased survivin expression might result in
embryotoxic effects, which was tested and validated using a set of
known non-embryotoxic (acrylamide), moderately embryotoxic
(tetracycline and boric acid), and strongly embryotoxic drugs
(retinoic acid, Ginkgo Biloba extract and methotrexate hydrate). It
was found that strongly embryotoxic drugs significantly decreased
survivin expression, while moderately embryotoxic drugs reduced
survivin expression slightly. Additionally, it was found that
acrylamide upregulated survivin expression due to its tumorigenic
effects. These results showed that the HTS assay could serve as
a reliable, sensitive and robust method to screen embryotoxic
potential of chemical compounds.
Biophysical & Biomolecular
Processes:
Protein Characterization Technologies –
Structure, Stability, and Dynamics
BIOT 111 – 8:50 a.m.
Finding the “perfect” lead: Fast and predictive methods for the
assessment of stability properties of therapeutic proteins
Hubert Kettenberger, [email protected]
Research and Early Development (pRED), Roche Pharmaceuticals,
Penzberg, Germany
Before the selection of a lead molecule from a series of lead
candidates, it is desirable to assess their physico-chemical
properties and their resistance to protein degradation in order
to avoid liabilities during further development and formulation.
Time and material constraints require fast and predictive methods
to identify the most promising candidate. Parameters of interest
include – among others - expressability, chemical stability as well
as solubility and solution behavior at high protein concentration.
A combination of in silico and in vitro methods is used to establish
a “developability profile” of protein candidates. The presentation
will focus on strategies and methods for early, small-scale stability
testing and implications for molecular design.
BIOT 112 – 9:10 a.m.
8:30 a.m. Room# 25B
J. Laurence, O. Stauch Papers 110-117
Investigation of the relationship between stability and
chemical modification in Fc-conjugates
BIOT 110 – 8:30 a.m.
Wen Zhou1, [email protected], Robert J. Proos1, Janet L
Wolfe1, Jennifer S Laurence2. (1) Wolfe Laboratories, Inc., Watertown, MA
02472, United States (2) Pharmaceutical Chemistry, University of Kansas,
Lawrence, KS 66047, United States
Design of antibodies specific for unfolded proteins
Ali Reza A Ladiwala, Joseph M Perchiacca, Moumita Bhattacharya,
Peter M Tessier, [email protected] of Chemical & Biological
Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180,
United States
Protein unfolding leads to aggregation and loss of function.
To monitor protein unfolding at low concentrations or in the
presence of other folded proteins, we aim to develop antibodies
specific for unfolded proteins. We posit that antibodies specific for
solvent-exposed aromatic residues (which are normally solventshielded within folded proteins) will selectively recognize unfolded
conformations of diverse proteins. Based on this hypothesis, we find
that the complementarity determining regions of small antibodies
can be engineered to selectively recognize unfolded proteins via pistacking interactions. We will discuss how these novel antibodies
can be used to monitor protein unfolding in a highly sensitive and
site-specific manner.
Antibody-drug conjugates (ADCs) hold great promise to
specifically deliver cytotoxic drugs to targets such as tumor
cells, and as such, have become one of the fastest growing
class of therapeutic agents. Covalent attachment of an organic
cytotoxin to a monoclonal antibody, however, alters the stability
of the protein conjugate. Despite the importance of this for
manufacturing, processing, and storage of ADCs, the influences
of conjugation on the physical stability of the protein have not
been widely or systematically studied. Here, we present our
investigation of a systematically modified Fc region of IgG1 using
a set of fluorophores and characterize the relationship between
the protein’s physical stability and hydrophobicity and/or surface
charge. The fluorophores were conjugated to Fc at Lys residues
and the conjugates were characterized using chromatographic
methods, including size-exclusion chromatography, ion exchange
chromatography, and hydrophobic interaction chromatography,
as well as differential scanning calorimetry, light scattering, and
circular dichroism.
55
Monday Morning
BIOT 106 – 10:10 a.m.
BIOT 115 – 10:30 a.m.
Escherichia coli skp chaperone sequesters kinetically trapped
intermediates of soluble proteins to reduce aggregation
Predicting solvent effects on protein conformations by
complete characterization of the protein solvation mosaic
Kevin C Entzminger, [email protected], Christine Chang, Ryan
Myhre, Jennifer A Maynard.Department of Chemical Engineering,
University of Texas at Austin, Austin, TX 78712, United States
Vincent Vagenende1, [email protected],
Bernhardt L Trout2. (1) Bioprocessing Technology Institute (A*STAR),
Singapore, Singapore 138668, Singapore (2) Department of Chemical
Engineering, Massachusetts Institute of Technology, Cambridge,
The periplasmic skp chaperone has been characterized primarily
for its role in outer membrane protein (OMP) biogenesis, in
which the jellyfish-like trimeric protein encapsulates partially
folded OMPs until delivery to the outer membrane. However,
skp is being increasingly recognized as a mediator of soluble
protein folding; skp co-expression increases the active yields of
many recombinant proteins and bacterial virulence factors. The
molecular mechanisms governing soluble protein recognition by
skp are currently unknown. Using a panel of single-chain variable
fragments (scFvs), we performed in vitro folding and aggregation
assays in the presence or absence of skp. Kinetic data for skpsensitive scFvs were fitted globally to a model whereby skp acts to
reduce aggregation by sequestering partially folded intermediates.
Folding intermediates were further confirmed by equilibrium
denaturation. These results will aid future engineering projects
aimed at achieving high-level recombinant protein yields in E. coli
and identify novel approaches to block bacterial virulence.
Massachusetts 02139, United States
BIOT 114 – 10:10 a.m.
What color is your protein? Using a quantitative color
assessment method
Limitations in computational resources and the lack of suitable
experimental techniques to probe protein solvation in mixed
solvents - which comprises a mosaic of preferentially hydrated,
solvated and neutral solvent regions - have restricted current
understanding of solvent effects on proteins. To the best of our
knowledge, this is the first study that has characterized the solvation
mosaic over the entire protein surface. This is achieved for two
proteins, lysozyme and an antibody fragment, in a mixture of water
and glycerol by the rigorous statistical analysis of microsecond allatom molecular dynamics simulations. We find that conformational
changes of single side-chains can alter local protein solvation
and we predict the solvent-induced population-shift of protein
conformations. Our methodology for characterizing the solvation
mosaic is applicable for a wide range of cosolvents and proteins,
and hold broad potential for advancing understanding of solvent
effects on protein processes.
BIOT 116 – 10:50 a.m.
Nonideality in high concentration solutions
Trevor E Swartz1, [email protected], Tom Patapoff1, Bruce
Ronald Toth, [email protected], Susan Chase, Thomas Laue.
Currently a visual assessment is used for color characterization of
liquid formulation drug product. This involves an analyst finding
the best fit of a sample to a color series. This method, based on the
European Pharmacopia, is inherently subjective because it requires
one or more analyst to make a judgment of the best match of a sample
to a color series. This talk will describe how information about the
color of a solution is captured in the visible absorption spectrum.
This spectrum can then be converted to a color space which allows
for color series matching to be performed in a quantitative way.
This quantitative method can be employed for both low and high
concentration liquid formulations. The effect of impurities on the
color assessment of high and low concentration formulations will
also be presented. This method also allows for predicting the relative
color of samples after a single chromatography purification step.
Our current understanding of molecular interactions is based
mostly on research conducted in dilute solutions. In vivo biological
systems differ significantly from dilute solutions. The excluded
volume theory has been developed to describe such solutions.
The following will present evidence as to whether this theoretical
framework is sufficient to explain experimental data from solutions
with macromolecular concentrations up to 70 grams per liter.
Sedimentation velocity and equilibrium experiments were
conducted on green fluorescent protein as a tracer in high
concentrations of 3 crowding molecules, hen egg-white lysozyme,
dextran, and soybean trypsin inhibitor in the FDS optical system.
All solutions were made in phosphate buffered saline pH 7.4.
In the equilibrium experiments, GFP in HEL was found to have
a high degree of attractive nonideality, due to charge-charge
attraction between GFP and HEL. GFP in STI and GFP in dextran
were found to have a similar degree of repulsive nonideality. In the
Kabakoff1, Jian Yin1, Sarah Du2, Kimia Rahimi2, John De Los Santos2.
(1) Early Stage Pharmaceutical Development, Genentech, South San
Francisco, CA 94080, United States (2) Protein Analytical ChemistryTesting, Genentech, South San Francisco, CA 94080, United States
56
Department of Biochemistry, University of New Hampshire, Durham, New
Hampshire, United States
interference experiments, dextran was found to have an extremely
high effective volume in solution, explaining the similarity in
repulsive nonideality between GFP in STI and GFP in dextran.
Additionally, dextran alone had a greater degree of repulsive
nonideality than did GFP in dextran, pointing towards attraction
between GFP and dextran.
In the sedimentation velocity experiments, GFP was found to have
significant interactions with both HEL and dextran. A variety of
excipients was added to determine the nature of the interaction.
The only excipient seen to eliminate the second peak was NaCl.
This points to charge-charge attraction between GFP and dextran.
Such unexpected results point towards additional complications in
high concentration solutions that excluded volume theory does not
account for. Additional study and the development of a new model
are necessary to fully understand nonideality in high concentration
solutions.
Monday Morning
BIOT 113 – 9:30 a.m.
BIOT 117 – 11:10 a.m.
Factor VIII activation studied by HX-MS
Johan H Faber, [email protected], Mette Dahl Andersen, Anders
Svensson, Ole Hvilsted Olsen, Henning R Stennicke.Protein Structure and
Biophysics, Novo Nordisk A/S, Maaloev, Denmark
Factor VIII (FVIII) is a pro-cofactor that plays a critical role in an
intermediate step in the blood coagulation cascade. Mutant forms
of FVIII or FVIII deficiencies are the cause of hemophilia A. The
protein consists of a heavy chain and a light chain composed of
covalently linked subunits A1-A2-B and A3-C1-C2, respectively.
Factor VIII may be activated by proteolytic cleavage catalyzed by
thrombin.
In this study, we apply Hydrogen/deuterium exchange (HX)
detected by mass spectrometry (MS) analyses on the conformational
states of recombinant human FVIII and its thrombin activated
form FVIIIa. 255 peptic peptides were used for analysis providing
93% sequence coverage of the 170 kDa non-redundant FVIII. The
HXMS structural characterization analyses reveals strong similarity
between FVIII and FVIIIa. Apart from indications of lowered
interactions between the two chains of FVIIIa upon thrombin
activation, no major conformational changes are observed.
57
A
B
C
D
E
Room# 20 B/C
Company Lunch Seminar hosted by GE Healthcare Biotechnologies
“New Developments in Single-Use Bioprocess Control and Sensing Technology”
2:00 p.m.
Room# 16A
Downstream Processes: Antibodies and Antibody-like Molecules
A.Ubiera, J. Neville Papers 118-125
2:00 p.m. 2:00 p.m.
Room# 16A
A.Ubiera, J. Neville Papers 118-125
2:00 p.m Room# 16B
S. Garneau-Tsodikova, S. Ma Papers 126-132
Room# 17A
Advances in Biofuels Production: Applications of Systems Biology,
Synthetic Biology, and Metabolic Engineering
H. Alper, S. Atsumi, C. Trinh Papers 133-140
2:00 p.m.
Room# 25A Stem Cells and Tissue Engineering: Mechanisms and Models of Stem
Cell Fate Determination
B. Rao, U. Lakshmipathy Papers 141-148
2:00 p.m. Room# 25B
Biophysical & Biomolecular Processes: Protein Characterization
Technologies – Interactions and Assembly
Y. Gorkarn, P. Tessier Papers 149-156
5:00 - 6:00 p.m.
6:00 -10:00 p.m.
6:00 p.m.
The David Perlman Award Lecture
Executive Committee Meeting Sci-Mix BIOT 118 – 2:00 p.m.
Evaluation of alternative cleaning strategies for protein A
chromatography adsorbents
Meisam Bakhshayeshi1, [email protected],
Bob Chen1, Ionela Iliescu1, Justin McCue1, Susanne Wood2. (1) Process
Biochemistry, Biogen Idec, Cambridge, MA 02142, United States (2)
Downstream Development, Biovitrum AB, Stockholm, Sweden
Upstream Processes: Engineering Natural Products Biosynthesis
Antibodies and Antibody-like Molecules
Presenter: Timothy Wortley
58
12:30 -2:00 p.m.
Downstream Processes:
Bill Rastetter Room#16A
Room# 26A
Hall D
Protein A chromatography has become an integral part of the
purification processes for recombinant therapeutics. The protein
A capture step is typically used for purification of monoclonal
antibodies and Fc fusion proteins. Due to the high cost of protein
A chromatography adsorbents, the adsorbent must be used for
numerous cycles during the manufacturing process to decrease
the cost of goods. When used for many cycles, it is critical that
the adsorbent provides consistent process performance and
product quality over the adsorbent lifetime. The objective of this
study was to evaluate different cleaning approaches for a protein
A chromatography adsorbent in scale down columns. Effective
cleaning of the protein A absorbent used for purification of a Fc
fusion protein was initially found to be very challenging using
a conventional cleaning strategy. We evaluated the lifetime of
the Protein A adsorbent using several different regeneration
conditions. The regeneration conditions included the use of
different concentrations of acid and base solutions, used in different
degrees of frequency during column cycling studies.
As part of the performance evaluation, the trends in adsorbent
capacity, step yield, product quality, and product carryover were
evaluated and monitored over the course of the cycling studies.
As part of the study, the mechanisms responsible for changes in
column performance were also evaluated, including protein A
ligand leachate loss, and changes to adsorbent mass transport
properties as a result of insufficient cleaning. As a result of this
study, we were able to identify cleaning conditions which were
able to properly balance cleaning efficiency with column lifetime,
and were able to extend the column lifetime accordingly. The
results provide important insights into the optimum regeneration
strategies for protein A chromatography media.
BIOT 119 – 2:20 p.m.
Characteristics of host cell protein removal during Protein A
chromatography
Richard D R Tarrant1, [email protected], Andrew
S Tait1, C Mark Smales2, Daniel G Bracewell1. (1) Department of
Biochemical Engineering, University College London, London, London
WC1E 7JE, United Kingdom (2) School of Biosciences, University of Kent,
Canterbury, Kent CT2 7NJ, United Kingdom
Protein A chromatography remains a critical step in the
purification of mAb and related products. Its ability to remove
>98 % of impurities alleviates the burden on subsequent steps and
facilitates platform processing. We have evaluated the ability of
four commercially available Protein A chromatography matrices
to remove host cell proteins (HCPs), based on a scale-down
process for an IgG4 produced in CHO cells. SELDI-TOF mass
spectrometry was used to generate a contaminant profile for each
resin and demonstrated that a number of components are shared,
but further peaks are associated with porous glass. To further
examine the behaviour of these contaminants a null cell line
approach was used to prepare enriched samples of HCPs for 2D-gel
electrophoresis. Some contaminants are associated with the resin
backbone while others with the product. This characterisation may
provide a rationale for targeted process development or cell line
engineering strategies to remove challenging contaminants.
BIOT 120 – 2:40 p.m.
Novel cyclic peptide ligand for antibody purification identified
by screening an mRNA-display library of cyclic peptides
Stefano Menegatti2, [email protected], Mahmud
Hussain2, Amith D. Naik2, Balaji M. Rao2, Ruben G. Carbonell1,2. (1)
Biomanufacturing Training and Education Center (BTEC), North Carolina
State University, Raleigh, North Carolina 27695, United States (2)
Department of Chemical and Biomolecular Engineering, North Carolina
State University, Raleigh, North Carolina 27695, United States
Monoclonal antibodies are an important class of therapeutics for
the treatment of severe diseases. The manufacturing of antibodybased drugs involves the use of Protein A and Protein G media,
which are expensive and suffer from low chemical stability. To
overcome these limitations, novel synthetic molecules have been
proposed as affinity ligands. In particular, cyclic peptides are
very promising candidates due to their high target specificity and
biochemical stability. Recently we have identified a cyclic peptide
that selectively binds the Fc region of antibodies by screening
an mRNA-display library of cyclic peptides prepared by peptide
cyclization on a “reversible solid-phase” format. The cyclic peptide
was synthesised on a chromatographic resin applying a novel
59
Monday Afternoon
Monday Afternoon
Sessions
Monday Afternoon Sessions
BIOT 121 – 3:00 p.m.
Identification and characterization of host cell protein
product-associated impurities in monoclonal antibody
downstream processing
Nicholas E Levy, [email protected], Kristin Valente, Kelvin H Lee,
Abraham M Lenhoff.Department of Chemical Engineering, University of
Delaware, Newark, DE 19716, United States
Downstream purification of mAbs has evolved to allow the
specific process for a new product to be developed largely by
empirical specialization of a platform process to allow removal of
impurities of different kinds. A more fundamental understanding
of impurities and the product itself would provide insights into
the rational design of efficient downstream processes. This work
identifies and characterizes host cell protein (HCP) productassociated impurities, i.e., HCP species carried through the
downstream processes via interactions with the mAb. Interactions
between HCP and mAbs are characterized using cross-interaction
chromatography over a range of conditions typical of those used in
downstream processing. The interacting species are then identified
by two-dimensional gel electrophoresis and mass spectrometry.
This methodology has been applied to identify product-associated
impurities in one particular purification step, namely protein A
affinity chromatography, for a variety of therapeutic mAbs. The
results show both the variation in HCP-mAb interactions with
different mAbs, and the relative importance of product-association
in protein A affinity chromatography.
BIOT 122 – 3:40 p.m.
Surface induced denaturation of monoclonal antibodies on
cation exchange chromatographic media
Ronald O. Gillespie1, [email protected], Sean MacNeil1,
Thao Nguyen1, Laurie Jones2, Shon Crampton3, Suresh Vunnum1. (1)
Department of Purification Process Development, Amgen Inc, Seattle,
Washington 98119, United States (2) Department of Analytical Sciences,
Amgen Inc., Seattle, Washington 98119, United States (3) Department
of Drug Product Development, Amgen Inc., Seattle, Washington 98119,
United States
Recent observations with a deglycosylated monoclonal antibody
have confirmed that mAbs are susceptible to surface induced
denaturation even in mild chromatographic systems, such as ion-
60
exchange1,2. Denaturation in that case was attributed to decreased
stability associated with the lack of glycosylation. An extension
of that work demonstrated that surface induced denaturation on
ion-exchange media is more common than previously thought
even for glycosylated mAbs. Surface induced denaturation can
manifest as tailing peaks or peak splitting and significant aggregate
generation, resulting in difficulties in achieving targeted product
quality attributes. Examples of several mAbs prone to CEX induced
denaturation will be presented and the impact of CEX operating
variables on the extent of denaturation will be discussed in detail.
Mitigation strategies for the continued use of this unit operation
for impurity removal without impacting product integrity will also
be discussed.
1.
Gillespie et al. “Chromatographic surface induced
denaturation of mAbs: Challenges for purification development”,
2010, 239th ACS National Meeting, San Francisco, CA
2.
Diao et al. “Characterization and investigation of the
double-peak cation exchange chromatography elution profiles of
an aglycosylated monoclonal antibody”, 2011, PREP conference,
Boston, MA
BIOT 123 – 4:00 p.m.
Implementation of membrane chromatography into
continuous antibody purification process
Ying Hou, [email protected], Mark Brower, Alexandra Buttke,
David J Pollard.Biologics-New and Enabling Technologies, BioProcess
Development, Merck & Co., Inc., Rahway, NJ 12180, United States
The production of monoclonal antibodies (mAbs) demands
economical downstream purification process with high purity, yield,
and throughput. In contrast to traditional batch based operations,
continuous processing through the various purification steps (or
downstream unit operations) offers a number of advantages. These
include faster overall processing time, lower cost and less storage
facilities. In this work, a continuous mAbs purification process has
been developed with the use of emerging technologies and singleuse, disposable products, such as single-use centrifugation and
simulated moving bed (SMB) chromatography. A single-use anion
exchange membrane chromatography step was developed to be
part of this continuous process because of its high binding capacity
and reduced buffer consumption compared to the traditional
batch based process. A case study using this continuous process is
discussed and results show significant productivity improvement
with the same product quality as compared to batch operation
mode.
BIOT 124 – 4:20 p.m.
Hydrophobic interaction as an effective conditioning step for
parvo virus filtration feed streams
Björn Hansmann1, [email protected], Anika
Meyer2, Nathalie Frau3, Volkmar Thom1. (1) Membrane R&D, Sartorius
Stedim Biotech GmbH, Göttingen, Germany (2) Product Management,
Sartorius Stedim Biotech GmbH, Göttingen, Germany (3) Process R&D,
Sartorius Stedim Biotech GmbH, Göttingen, Germany
Foulants, often present in trace amounts only, can severely limit
virus filter capacity and throughput. Adsorptive prefiltration
can circumvent premature blocking and significantly increase
virus filter performance. An adsorptive prefilter membrane is
presented. It binds foulants by hydrophobic interactions and
is relatively unaffected by feed stream changes in pH and shows
robust performance even at high ionic strength. It exhibits a low
extractables profile and can be autoclaved. Being a 0,1µm pore size
membrane, it concomitantly acts as a size exclusion guard filter
membrane and is integrity testable. The performance increase in
virus filtration capacity with adsorptive prefiltration is shown for
different feed solutions and compared to alternative prefiltration
media. Binding isotherms for model foulants as a function of
pH and ionic strength are presented. An attempt is made, to
fundamentally explain the fouling of virus filters and the respective
action of adsorptive prefilters. Additional data describes the
interaction of the prefilter material with detergents often present
in final formulations.
Upstream Processes:
Engineering Natural Products Biosynthesis
2:00 p.m Room# 16B
S. Garneau-Tsodikova, S. Ma Papers 126-132
BIOT 126 – 2:00 p.m.
Tools for natural product genome mining of microbioal
metabolic exchange
Peter Dorrestein, [email protected] of California,
San Diego, United StatesDepartments of Pharmacology, Chemistry and
Biochemistry, Skaggs School of Pharmacy and Pharmaceutical Sciences,
United States
In this lecture, the development of mass spectrometry tools by
our laboratory will be described that enable the detection and
characterization of natural products in 3D as well as directly
from living microbial colonies. These tools, in conjunction with
molecular networking approaches for genome mining and flux
analysis, provide exciting opportunities to directly link microbial
phenotypes with genotypes and chemotypes. Applications of these
tools are broad reaching ranging from antimicrobial discovery,
the identification of factors involved in the protection of crops,
the identification of the chemistry involved in symbiotic relations
between plants or coral and microbes, and how our microbiome is
involved in the control of infections or development of dandruff.
BIOT 125 – 4:40 p.m.
Comparison of adsorbtive membranes to traditional anion
exchange chromatography as an alternative purification step
for monoclonal antibodies
George Miesegaes, [email protected], Scott Lute, Erik
Read, Jessica Dement-Brown, Kurt Brorson.Center for Drug Evaluation
and Research, US Food and Drug Administration, Silver Spring, MD
20901, United States
Membrane adsorption chromatography has become a popular
downstream purification step for monoclonal antibodies. This
technology offers advantages such as disposability, high capacity,
and ease of use. Mechanistically, adsorbers are believed to function
similar to flow-through mode anion exchange chromatogarphy. In
this scenariom the electrostatic environment is such that the desired
product of interest passes through the media, while undesired
contaminants do not. We performed a series of experiments to
assess the comparability of adsorbers with anion exchange. Using a
model antibody feedstock, we assed the ability of both operations
at small-scale to retain host cell DNA and protein, and viruses.
BIOT 127 – 2:20 p.m.
Harnessing the native substrate flexibility of biosynthetic P450
enzymes for natural product structural diversification
Shenging Li1, [email protected], Jacob C. Carlson1, Karoline
Chiou1, Mani Raj Charlagain2, Allison R. Knauff2, Shamila S.
Gunatilleke3, Yojiro Anzai1, Douglas A. Burr1, John Montgomery2,
Larissa M. Podust3, David H. Sherman1. (1) Department of Life Sciences,
University of Michigan, United States (2) Department of Chemistry,
University of Michigan, United States (3) Departments of Pathology and
Sandler Center for Drug Discovery, University of California, San Francisco,
United States
Diverse cytochrome P450 monooxygenases (CYPs) are involved
in an expansive array of regio- and stereoselective oxidations in
the biosynthesis of natural products. Individual CYPs with native
substrate flexibility hold particular potential for development
into biocatalysts to selectively oxidize complex natural product
and synthetic substrates, thus leading to structurally diverse
products with enhanced bioactivity. The PikC1 and TamI2 CYP
61
Monday Afternoon
method devised to replicate the peptide composition and structure
as on the mRNA-peptide hybrid. The resulting affinity adsorbent
was used to recover IgG from a cell culture medium with yield and
purity up to 90% and 95%.
spirolactone. We confirmed the tryptoquialanine pathway went
through an intermediate common to the fumiquinazoline pathway,
fumiquinazoline F, which originates from a fungal trimodular
nonribosomal peptide synthetase (NRPS). We also established the
biosynthetic sequence of the pathway by systematically inactivating
single gene and characterization of the intermediates. An unusual
oxidative opening of the pyrazinone ring by an FAD-dependent
oxidoreductase has been proposed. Notably, a 2-aminoisobutyric
acid (AIB)-utilizing NRPS has been identified and reconstituted in
vitro, along with two putative enzymes of unknown functions that
are involved in the synthesis of AIB by genetic analysis.
interpolypeptide ACP:KS interactions in polyketide and fatty
acid synthases with single-residue resolution. A high-throughput
format will allow probing protein-protein interactions for directed
evolution. Prosthetic arms of polyketide biosynthetic machinery
(phosphopantetheine, Ppant) are installed via post-translational
modification onto acyl carrier proteins, and play an intimate role
in PKS specificity and catalysis. We are installing a broad array of
Ppant analogs via unnatural amino acid mutagenesis and click
chemistry. Ultimately, non-natural analogs that broaden the
substrate promiscuity of domains in natural product biosynthesis
will be identified.
BIOT 130 – 3:40 p.m.
BIOT 132 – 4:40 p.m.
Texas at Austin, United States
Genomics-inspired discovery of natural product chemistry
Role of leader peptides in lasso peptide biosynthesis
Chiral building blocks are useful starting materials in the syntheses
of natural products and pharmaceuticals. Our lab has been
employing isolated enzymes from modular polyketide synthases
as well as auxiliary enzymes as biocatalysts to generate desired
diketide and triketide building blocks. Provided with simple
precursors and methylmalonyl extender units generated by a
malonyl-CoA ligase, engineered polyketide synthase modules are
capable of synthesizing valuable polyketides. These enzymes are
remarkably regio- and stereospecific catalysts even in these in vitro
systems and can be employed to inexpensively produce preparative
quantities of desired stereopure products, thereby establishing a
new paradigm in green chemistry.
Bradley Moore, [email protected] Institution of
Oceanography and Skaggs School of Pharmacy and Pharmaceutical
A. James Link, [email protected] Chemical and
BIOT 128 – 2:40 p.m.
Employing polyketide synthase enzymes as biocatalysts to
generate chiral building blocks
Adrian Keatinge-Clay, [email protected] of
BIOT 129 – 3:00 p.m.
Fungal indole alkaloid biosynthesis: Genetic and biochemical
investigation of the tryptoquialanine pathway in Penicillium
aethiopicum
Xue Gao1, [email protected], Yit-Heng Chooi1, Brian D. Ames3,
Christopher T. Walsh3, Yi Tang1,2. (1) Department of Chemical and
Biomolecular Engineering, University of California, Los Angeles, Los
Angeles, CA 90095, United States (2) Department of Chemistry and
Biochemistry, University of California, Los Angeles, Los Angeles, CA
90095, United States (3) Department of Biological Chemistry & Molecular
Pharmacology, Harvard Medical School, Boston, Massachusetts 02115,
United States
Tryptoquialanine is a quinazoline-containing fungal indole
alkaloid and highly similar to the known tremogen tryptoquivaline.
These tremogenic compounds are capable of eliciting intermittent
or sustained tremors in vertebrate animals. Here, we report the
characterization of the tryptoquialanine biosynthetic pathway,
including the formation of the acetylated quinazoline ring
connected to a 6-5-5 imidazoindolone ring system via a 5-membered
62
Sciences, United States
Natural product compounds have historically been discovered
based on their chemical or biological properties. With the ease
and affordability of genome sequencing today, a new era in
natural product discovery is unfolding in which genomics and
biosynthesis are driving new innovations in compound discovery
based on the mining of genomic information. This orthogonal
discovery approach takes advantage of the biosynthetic potential
of a genome-sequenced organism to design hypothesis-driven
experiments to uncover new chemical entities. Examples from
the author’s laboratory will highlight the myriad of available and
evolving genome mining approaches to new natural product
chemistry and biosynthetic enzymology.
BIOT 131 – 4:20 p.m.
Biological Engineering, Princeton University, Princeton, New Jersey 08544,
United States
Lasso peptides are a class of ribosomally-synthesized natural
products that are attractive as drug targets because of their extreme
stability, sequence diversity, and ability to be engineered using
protein engineering techniques. As is the case for many ribosomal
natural products, a precursor protein is matured into the final
product via posttranslational modifications. This precursor
protein contains an N-terminal leader peptide segment followed
by the C-terminal core peptide region. There is a low degree of
sequence conservation across different lasso peptide precursors,
and the length of these precursors varies widely. In this talk I will
discuss our work in elucidating the function of leader peptide in
the biosynthesis of the lasso peptides microcin J25 and capistruin. I
will address how this information enables us to discover new lasso
peptides from genomic sequence data.
Advances in Biofuels Production:
Applications of Systems Biology, Synthetic Biology, and Metabolic Engineering
2:00 p.m. Room# 17A
H. Alper, S. Atsumi, C. Trinh Papers 133-140
BIOT 133 – 2:00 p.m.
Genome-scale model and pathway analysis for optimization of
butyric acid fermentation by Clostridium tyrobutyricum
Ying Jin, [email protected] of Chemical and Biomolecular
Engineering, The Ohio State University, Columbus, Ohio 43210,
United States
Understanding the metabolic behavior of an organism or a
living cell is essential for fermentation process. Predictions of
metabolic behavior and flux distribution in batch cultures enable
better regulation of fermentation in terms of biomass yield and
productivity. With the increasing amount of biological knowledge,
from genome to metabolome, it becomes relatively straightforward
to exploit metabolic and regulatory pathways for fermentation
optimization. In this study, a genome-scale model was developed
using Pathway Tool and used for systematical analysis of butyric
acid fermentation by Clostridium tyrobutyricum. The model was
used to study the metabolic pathway in this organism and perform
flux analysis to understand the constraint factors of fermentation,
optimize the process and improve product yield. The results from
this analysis will be presented in this paper.
New chemical biology approaches for engineering natural
product assembly lines
BIOT 134 – 2:20 p.m.
Zhixia Ye1, [email protected], Morgan Blair1,2, Hemant Desai1, Gavin
Comparison of microbial metabolic networks to guide
background strain selection
Williams1. (1) Department of Chemistry, North Carolina State University,
Raleigh, North Carolina 27695, United States (2) Department of
Chemistry, Berry College, Mount Berry, GA 30159, United States
We are using chemical biology strategies to improve the scope
and efficiency of combinatorial polyketide biosynthesis. We
focus on two crucial aspects, protein:protein interactions and
prosthetic arm engineering. Understanding interpolypeptide
ACP:KS communication is crucial to improve the outcome of
combinatorial biosynthesis aimed at module swapping to generate
polyketide analogues. A para-benzoyl-L-phenylalanine (pBpa)
based photocrosslinking approach has been employed to map
Joshua J Hamilton, [email protected], Jennifer L Reed.
Department of Chemical and Biological Engineering, University of
Wisconsin-Madison, Madison, WI 53706, United States
Genome-scale metabolic reconstructions are the foundation
for computational methods to understand and improve cellular
phenotypes. While many such methods have been developed, they
are generally only applied to models of single organisms. Existing
methods for comparing multiple organisms via their network
reconstructions can identify reconstruction differences, but not
63
Monday Afternoon
enzymes involved in biosynthesis of the antibiotics pikromycin
and tirandamycin, respectively, possess this type of natural
substrate flexibility. Detailed biochemical, crystallographic,
and bioengineering studies have enabled us to understand and
manipulate their unique substrate recognition mechanism to
expand the oxidative product profile resulting in a new spectrum
of bioactive molecules
BIOT 135 – 2:40 p.m.
Next generation RNA-seq based transcriptomics on the
microalgae Neochloris oleoabundans: Implications for
improved lipid biosynthesis
Berat Z Haznedaroglu1, [email protected], Hamid
Rismani-Yazdi2, Jordan Peccia1. (1) Department of Chemical and
Environmental Engineering, Yale University, New Haven, CT 06511, United
States (2) Department of Chemical Engineering, Massachusetts Institute of
Technology, Cambridge, MA 02139, United States
Limitedly available genome sequences for nonmodel microalgae
as promising candidates for fuel production preclude the adoption
of a rational approach to metabolic engineering-based biofuel
feedstock optimization studies. To fulfill this gap of knowledge, we
described the nucleic acid sequencing and de novo transcriptome
assembly of lipid enriched microalgae Neochloris oleoabundans, in
which we successfully identified and mapped genes and pathways
of importance for biofuel production including triacylglycerol
(TAG) and fatty acid biosynthesis. Our RNA-seq results also
indicate that environmental triggers for TAG synthesis also result
in the upregulation of enzymes responsible for lipid degradation
in lysosomes and spliceosomes, demonstrating the importance
of a systems biology approach to understand the mechanisms of
improved lipid synthesis. Results to be presented at the 243rd ACS
National Meeting will include the constructed metabolic pathways
involved in the biosynthesis and catabolism of fatty acids in N.
oleoabundans as well as the assembled transcriptome which will
provide a foundation for further analysis of molecular genetics
and functional genomics required to direct metabolic engineering
efforts to enhance both quality and quantity of microalgae-based
biofuel feedstock.
BIOT 136 – 3:00 p.m.
Elucidating and optimizing E. coli metabolism for obligate
64
anaerobic isobutanol and butanol production
Cong T Trinh, [email protected] and Biomolecular
Engineering, University of Tennessee, Knoxville, Knoxville, TN 37996,
United States
Elementary mode (EM) analysis was applied to elucidate and
compare E. coli fermentative metabolisms for obligate anaerobic
production of butanol and isobutanol. The result shows that
butanol fermentative metabolism was NADH-deficient while
isobutanol fermentative metabolism was NADH-redundant. E.
coli could grow and produce butanol anaerobically as the sole
fermentative product but not achieve maximum butanol yield.
In contrast, for isobutanol fermentative metabolism, E. coli was
required to couple with either ethanol- or succinate-producing
pathway to recycle NADH. To overcome these “defective”
metabolisms, EM analysis was implemented to redesign native E.
coli fermentative metabolisms for optimized anaerobic production
of butanol and isobutanol. Even though butanol and isobutanol
fermentative metabolisms were quite different, designed strains
could be engineered to have identical metabolic flux distribution
in “core” metabolic pathways. We will compare experimental
data and model prediction in elucidating and redesigning E. coli
fermentative metabolisms for obligate anaerobic production of
butanol and isobutanol.
BIOT 137 – 3:40 p.m.
Conversion of proteins into biofuels: Toward nitrogen neutral
biofuel production
Yixin Huo1, [email protected], Kwang myung Cho1, James C.
Liao2. (1) Easel BIotechnologies, LLC, Los Angeles, CA 90025, United
States (2) Chemical Engineering, UCLA, Los Angeles, CA 90024,
United States
Current biofuel processes and developing approaches close the
carbon cycle with sequestration of atmospheric carbon dioxide,
but still rely on the fertilizer produced by the Haber-Bosch process.
A heretofore unexplored solution to these limitations would be to
use proteins as a feedstock. Proteins are the major component of
the photosynthesis apparatus, CO2 fixation pathways and other
biosynthetic and cell growth machinery. Thus, using proteins as
feedstock might maximize growth and CO2 fixation rates. Proteins
have not been used to synthesize fuels because of the difficulties of
deaminating protein hydrolysates. In this study, we apply metabolic
engineering to generate Escherichia coli that can deaminate protein
hydrolysates, enabling the cells to convert proteins to C4 and C5
alcohols at 56% of the theoretical yield. We show that common fast
growing species can be used as protein sources, producing up to
4,035 mg/l of alcohols from biomass containing ~22 g/l of amino
acids.
BIOT 138 – 4:00 p.m.
Metabolic engineering of cellulolytic Clostridia for n-butanol
production from lignocellulosic biomass
Xiaorui Yang, [email protected], Shang-Tian Yang.
Department of Chemical and Biomolecular Engineering, The Ohio State
University, Columbus, Ohio 43210, United States
Cellulose is the most abundant biomass on the earth. Butanol
production from lignocellulosic biomass by fermentation has been
considered to have great economic and environmental benefits.
Some Clostridia, including Clostridium cellulolyticum and
Clostridium cellulovorans, are able to use cellulose directly, but not
able to produce butanol. In this work, we aimed to engineer these
cellulolytic Clostridia to produce n-butanol directly from cellulose
by introducing novel n-butanol biosynthesis pathways. At the same
time, to reduce the acid byproducts, genes in lactic, acetic and
butyric acid pathways were knocked out to block their production.
With the engineered cellulolytic Clostridia, n-butanol was
produced directly from cellulose at a high productivity and yield,
making the process attractive for industrial application. This paper
will highlight different strategies applied in metabolic engineering
of cellulolytic Clostridia. The characterization and fermentation
results from these mutant strains will also be presented in this
paper.
BIOT 139 – 4:20 p.m.
Genomics-driven elucidation and construction of
combinatorial mutants for hydrolysate tolerance in
Escherichia coli
An important feature of robust biocatalysts will be their ability to
utilize renewable feedstocks, especially cellulosic biomass. We used
a novel genome-wide tool, TRackable Multiplex Recombineering
(TRMR), to evaluate changes of gene expression that confer
tolerance to hydrolysate, and acetate and furfural, two key
inhibitors of hydrolysate. TRMR comprises two libraries, “up” for
increased gene expression and “down” for decreased expression.
Individualized barcodes allow for microarray analysis. These
libraries contain single mutations for most genes in Escherichia
coli. We have identified single mutations resulting in tolerance, such
as the previously reported ahpC mutation, which improved growth
>200% in dilute hydrolysate. We combined acetate, furfural, and
hydrolysate data sets to visualize overlaps. Heirachercal clustering,
gene ontology, and regulatory and metabolic network analyses are
some methods used to identify targets from our genomics data. We
will present results from constructed combinatorial mutants and
highlight features of non-combinatorial beneficial mutations.
BIOT 140 – 4:40 p.m.
Utilizing the CoGeL (co-existing/co-expressing genomic
libraries) technology to develop the complex phenotype of high
tolerance to oxidative stress
Sergios A. Nicolaou, [email protected], Eleftherios T. Papoutsakis.
Department of Chemical and Biomolecular Engineering, University of
Delaware, Newark, DE 19711, United States
Industrial bioprocessing creates environments of heat, solvent, acid
and oxidative stress that can hinder cell production capabilities.
Resistant phenotypes are desired for growth or survival under
stress to improve product titers. Genomic libraries are often
screened for tolerant phenotypes, and we have developed the CoExisting/Co-Expressing Genomic Libraries (CoGeL) technology
and demonstrated its application in identifying genes imparting
higher acid tolerance in Escherichia coli (Nucleic Acids Research,
2011, doi:10.1093/nar/gkr817). Here, we focus on oxidative stress,
which is catastrophic to cells as it damages proteins, membranes
and DNA. CoGeL screening identified several resistant clones
including a genomic fragment that imparts higher survivability
under H2O2 stress. We interrogated the genes of this region by
examining individual gene knockouts. We identified the gene that
provides the main effect and demonstrated increased survivability
by gene overexpression. Aiming to provide mechanistic
understanding of this protection effect, the transcriptional effects
of the overexpressing insert were investigated.
Tirzah Y Glebes, [email protected], Nicholas R Sandoval,
Sophie J Weiss, Ryan T Gill.Chemical and Biological Engineering,
University of Colorado, Boulder, CO 80309, United States
65
Monday Afternoon
the effect these differences have on functional states of the network.
We have developed an approach to identify functional differences
between organisms, by finding conditions under which genetic
differences give rise to differences in metabolic capabilities.
We applied the algorithm to two existing reconstructions of
Escherichia coli, seeking metabolic engineering strategies that give
rise to different predicted biofuel production phenotypes. We were
subsequently able to identify differences in the reconstructions
responsible for the predicted production differences. We examine
the impact of these differences on common strain design strategies,
and discuss how this approach can guide selection of strains for
metabolic engineering.
Mechanisms and Models of Stem Cell Fate
Determination
2:00 p.m.
Room# 25A B. Rao, U. Lakshmipathy Papers 141-148
BIOT 141 – 2:00 p.m.
Multifactorial analysis of embryonic stem cell self-renewal
reveals a crucial role of GSK-3β-mediated signaling at
physiological oxygen levels
Hélder S. C. Barbosa, Tiago G. Fernandes, [email protected],
Tiago P. Dias, Maria Margarida Diogo, Joaquim M. S. Cabral.Department
of Bioengineering and Institute for Biotechnology and Bioengineering,
Center for Biological and Chemical Engineering, Instituto Superior Técnico,
Lisboa, Portugal
Work previously performed in our group showed that culturing
mouse embryonic stem (mES) cells under different oxygen
tensions gave rise to different cell proliferation patterns and
commitment stages dependent on which signaling pathways are
activated/inhibited to support mES cell self-renewal. However, a
clear understanding of the molecular mechanisms that regulate
stem cell fate and function under these conditions is mostly
lacking. To systematically investigate these effects, the sole and
interactive influence of MEK/ERK pathway inhibition, activation
of Wnt/β-Catenin and activation of STAT3 signaling, were
statistically evaluated during expansion of mES cells at different
oxygen tensions using a factorial design. This modeling approach
revealed that at lower O2 tensions STAT3 signaling and Wnt/βCatenin are indispensable for cell self-renewal and pluripotency.
Our results add new insights into the mechanisms by which
oxygen tension influences mES cell fate, and GSK-3β inhibition,
in particular, showed an important role towards maintenance of ES
cell pluripotency.
BIOT 142 – 2:20 p.m.
Trophectodermal differentiation of human embryonic
stem cells
Prasenjit Sarkar1, Timothy S Collier2, Shan M Randall2, David C
Muddiman2, Balaji Rao1, [email protected]. (1) Chemical and
Biomolecular Engineering, North Carolina State University, Raleigh, NC,
United States (2) Chemistry, North Carolina State University, Raleigh, NC,
United States
66
The placenta is a dynamically evolving organ that changes throughout
pregnancy and can be simplistically viewed as an interface between
the fetus and the mother where exchange of nutrients and waste
takes place, through an extensive system of villi. At all stages of
pregnancy, the placenta contains four important trophoblast cell
types – the villous cytotrophoblast (VCTB ), the multi-nucleate
syncytiotrophoblast (STB ), Extravillous Trophoblast Cell
Columns (EVT CC ) and the Invasive Extravillous Trophoblast
(I-EVT ). We have successfully obtained all key placental cell types
from hESCs. Our results show that, as in vivo, the development
of STBs, EVT-CCs and I-EVTs proceeds through a multipotent
VCTB intermediate. Here we present a mechanistic model for
differentiation of hESCs first to VCTBs and subsequently to STBs
and I-EVTs, as well as proteomic characterization of VCTBs and
VCTBs differentiating to I-EVTs.
BIOT 143 – 2:40 p.m.
Stem cell population heterogeneity: Development of a
quantitative framework
Jincheng Wu1, Emmanuel (Manolis) S. Tzanakakis1,2,3,
[email protected]. (1) Chemical and Biological Engineering, State
University of New York at Buffalo, Buffalo, NY 14260, United States (2)
New York State Center of Excellence in Bionformatics and Life Sciences,
State University of New York at Buffalo, Buffalo, NY 14203, United States
(3) Western New York Stem Cell Culture and Analysis Center, State
University of New York at Buffalo, Buffalo, NY 14214, United States
Human pluripotent stem cell (hPSC) populations are heterogeneous
hindering their efficient expansion and differentiation to desired
lineages. We developed a population balance equation model to
describe and predict the dynamics of human embryonic stem cell
(hESC) self-renewal. Intrinsic and extrinsic sources of population
heterogeneity as well as experimental data were considered in the
construction of the model. Solutions to the model were obtained
primarily by Monte Carlo methods. Model parameters pertinent to
cell growth, division and stem cell gene expression were calculated
from experiments. Additional experiments were carried out to
validate the simulation predictions. The contribution of intrinsic
sources considered here to hESC heterogeneity was found to be
almost 40%. Current efforts concentrate on expanding the model
to include additional sources of heterogeneity stemming from
hESC differentiation. The quantitative framework developed can
complement and accelerate investigations on stem cell propagation
or differentiation while reducing costly and labor-intensive
experimental procedures.
BIOT 144 – 3:00 p.m.
Nuclear rheostat that strongly couples microenvironment
rigidity to cell lineage
Dennis E Discher, [email protected], Joe Swift, Takamasa
Harada, Irena Ivanovska, Amnon Buxboim.Department of Chemical and
Biomolecular Eng’g., University of Pennsylvania, Philadelphia, PA 19104,
United States
A solid tissue can be soft like fat or brain, stiff like striated muscle
and heart, or rigid like bone. Proteomic profiling of tissue nuclei
shows that Lamin-A/C expression increases more than 30-fold and
in near-proportion to micro-elasticity of tissue, while other nuclear
envelope components such as Lamin-B exhibit small variations.
Lamin-A/C is absent in ESCs and has been implicated in aging
syndromes that affect muscle and fat but not brain. We find
nuclei in brain-derived cells are indeed dominated by Lamin-B
and are much softer than nuclei derived from muscle cells with
predominantly Lamin-A/C. In vitro, matrix elasticity can affect
expression of nuclear envelope components in adult stem cells, and
major changes in Lamin-A/C are indeed shown to direct lineage
with lower levels favoring soft tissue and higher levels promoting
rigid tissue lineage. At a molecular level, tagging of cryptic sites
while physically stressing isolated nuclei reveals stress-driven,
mass spectrometry-mapped changes in various nuclear proteins
including Lamin-A/C as well as chromatin-controlling proteins,
consistent with cell and tissue evidence that the nucleus transduces
physical stress.
BIOT 145 – 3:40 p.m.
Characterization of protein profile in cancer stem cells by
noncanonical amino acids
Xinrui Duan1, [email protected], Hongli Li1, Hong
Guan1, Hexin Chen2, Qian Wang1. (1) Department of Chemistry and
Biochemistry, University of South Carolina, Columbia, South Carolina
29208, United States (2) Department of Biological Sciences, University of
South carolina, Columbia, South Carolina 29208, United States
Many solid tumor types, including breast and colon cancer, are
found to contain small proportions of cells that are capable of
proliferation, self-renewal, and differentiation into the various cell
types seen in bulky tumors. These cells, termed “cancer stem cells”
(CSCs), may be responsible for treatment resistance and tumor
relapse. Several methods have been established to isolate CSCs from
bulky tumors, including suspension culture and surface markerbased cell sorting. Metabolic labeling of proteins with non-canonical
amino acids combing with fluorogenic probe provides powerful
tools to study spatial and temporal variation in protein profile. In
this work, the methionine analogue homopropargylglycine (HPG)
was used to metabolic labeling of new synthesized proteins in
isolated CSCs and non-CSCs. A fluorogenic probe reacted with
HPG after labeling by “Click chemistry” to provide visual signals.
Our study will help to identify difference between CSCs and nonCSCs in protein level in a spatial and temporal manner.
BIOT 146 – 4:00 p.m.
Polymeric substrate control of BMP2-engineered mesenchymal
progenitor cell differentiation
A.K. Bherwani1, [email protected], Chue-Cheng
Chang2, G Pelled3, Z. Gazit3, D. Gazit3, Miriam Rafailovich2, Marcia
Simon3. (1) Oral Biology/School of Dental Medicine, Stony Brook
University, Stony Brook, New York 11794, United States (2) Materials
Science, Stony Brook University, Stony Brook, New York 11794, United
States (3) Hadassah Faculty of Dental Medicine, Hebrew University,
Jerusalem, Israel
The differentiation of stem cells is controlled by multiple
cues received within different stem cell niche. To explore the
contribution of both surface mechanics and surface chemistry the
differentiation ofC3H10T1/2-derived progenitor cells genetically
engineered to express the differentiation inducer rhBMP-2, under
control of the Doxycycline (Dox)-repressible promoter, TetOff, was monitored following the growth of cells on cast films of
Polybutadiene (PB of 20nm and 200nm thickness) and partially
sulfonated polystyrene (PSS28) polymers and in the presence
or absence of doxycycline. Irrespective of differences in moduli,
evaluation by qPCR revealed that cells on PB exposed to BMP2
expressed markers of chondrogenesis (aggrecan), while cells on
PSS exposed to BMP2 expressed markers of osteogenesis (bone
sialoprotein). Additionally, cells on PSS generated biomineralized
deposits as determined by SEM and EDAX suggesting that surface
chemistry plays a significant regulatory role in endochondrial bone
formation.
BIOT 147 – 4:20 p.m.
Understanding differentiation mechanisms in retinoic acidtreated leukemic progenitor cells
Holly A Jensen1, [email protected], Jeffrey D Varner1, Andrew
Yen2. (1) Department of Chemical and Biomolecular Engineering, Cornell
University, Ithaca, NY 14853, United States (2) Department of Biomedical
Sciences, Cornell University, Ithaca, NY 14853, United States
Mechanisms underlying stem cell differentiation remain poorly
understood. The HL60 cell line has been a durable experimental
model since the late 1970s, and provides an archetype system for
investigating the intracellular networks that allow precursor stem
67
Monday Afternoon
Stem Cells and Tissue Engineering:
Biophysical & Biomolecular
Processes:
Protein Characterization Technologies –
Interactions and Assembly
2:00 p.m. Room# 25B
Y. Gorkarn, P. Tessier Papers 149-156
BIOT 149 – 2:00 p.m.
BIOT 148 – 4:40 p.m.
Utilizing protein-engineered biomaterials to create human
muscle tissue constructs
Debanti Sengupta1, [email protected], Penney M. Gilbert2,
Kyle J. Johnson3, Helen M. Blau2, Sarah C. Heilshorn3. (1) Department
of Chemistry, Stanford University, Stanford, CA 94305, United States
(2) Department of Microbiology and Immunology, Stanford University,
Stanford, CA 94305, United States (3) Department of Materials Science
and Engineering, Stanford University, Stanford, CA 94305, United States
Using recombinant protein technology, we synthesized a family of
protein-engineered biomaterials incorporating critical cues that
recapitulate in vivo muscle tissue conditions. These biomaterials
enable independent tuning of cell-adhesive ligand density and
topographical features. We are therefore able to decouple the effects
of biochemical and structural cues in achieving aligned, striated
muscle tissue using primary human skeletal muscle myoblasts
(hMBs) and adult muscle stem cells (MuSCs) isolated from tissue
biopsies. We utilized our biomaterial system to identify conditions
for the alignment and functional maturation of hMBs in order
to recreate the stem cell niche for MuSCs, which typically reside
atop mature muscle fibers. For our biomaterials, topographical
spacing of 20 microns and a cell-adhesive ligand density of
9,300 RGD ligands/micron2 enhanced primary hMB alignment,
elongation, and differentiation into multinucleated muscle fibers.
These engineered tissue constructs will enable studies of MuSC
niche requirements and may prove useful in regenerative medicine
therapies.
68
Biophysical, biochemical, and biological characterization
of two types of monoclonal antibody dimers: Role of protein
aggregate intermediates
Feng He1, [email protected], Christopher Woods1, Marisa Joubert2,
Edith Nalbanian1, Jonathan Woodard1, Pengzu Zhou3, Joey Pollastrini2,
Yinges Yigzaw1, Duke Phan1, Joanna Scavezze1, Christine Siska1, Thomas
Arroll1, Vibha Jawa4, Gerald Becker1, Linda Narhi2, Vladimir Razinkov1.
(1) Process & Product Development, Amgen, Seattle, WA 98119, United
States (2) Process & Product Development, Amgen, Thousand Oaks, CA,
United States (3) Small Molecule Process & Product Development, Amgen,
Thousand Oaks, CA, United States (4) Clinical Immunology,, Amgen,
Thousand Oaks, CA, United States
Aggregation has been identified as one of the major degradation
pathways that affect the quality and efficacy of protein therapeutics.
Dimers are the predominant oligomeric species found in
monoclonal antibody products, both during process and storage,
and following exposure to certain accelerated stress conditions.
While the dimers could be a heterogeneous population, it has been
hypothesized that a dimeric species could be the initial step on the
protein aggregation pathway. In this study, two dimer species were
isolated from monoclonal antibody samples upon long term storage
and elevated stress conditions. The dimer enriched fractions were
characterized for protein conformation, morphology, structural
integrity and bioactivity. The reversibility of dimers to monomers
was also assessed under various conditions. Furthermore, the
ability of dimers to enhance the early innate immune response
in a population of human peripheral blood mononuclear cells
was assessed in vitro by multiplex cytokine analysis. Distinct
cytokine patterns associated with the dimers were investigated
to determine their potential risk of immunogenicity. The results
revealed common properties and differences of the two types of
dimers generated under different physical conditions. The findings
of this study provide insights into the role of dimers in protein
aggregation pathway.
BIOT 150 – 2:20 p.m.
Predicting solvent effects on antibody binding affinity by
scrutinizing solvation at the binding interface
Vincent Vagenende1, [email protected],
Bernhardt L Trout2. (1) Bioprocessing Technology Institute (A*STAR),
Singapore, Singapore (2) Department of Chemical Engineering,
Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,
United States
Cosolvents such as urea, salts and polyols are commonly used in
protein processes, yet general understanding of solvent effects
on protein-protein interactions is lacking. Although the nature
of cosolvent-protein interactions is believed to be weak and
non-specific, addition of cosolvent strengthens protein-protein
interactions for some proteins, whereas it weakens proteinprotein interactions for others. This is exemplified by the puzzling
observation made years ago that glycerol increases the binding
affinity of one antibody, but decreases the binding affinity of a
second antibody to the same antigen. We have analyzed local protein
solvation at the binding interface of these antibodies by molecular
dynamics simulations and we present two key observations that
quantitatively explain the opposing effects of glycerol on the
antibody binding affinity. We show that our approach is generally
applicable for predicting solvent effects on the affinity of proteinprotein complexes for which atomic structures are available.
Plasmon wavelength) that is well correlated with light scattering
measurements obtained at orders of magnitude higher antibody
concentrations. We will present multiple examples of how SINS
can be used to optimize the formulation of therapeutic antibodies.
BIOT 152 – 3:00 p.m.
Assessing solution stability of pegylated proteins
Feng Jin, Kedar S. Deshpande, Marcel Ottens, m.ottens@tudelft.
nl.Biotechnology, Delft University of Technology, Delft, ZH 2628 BC, The
Netherlands
BIOT 151 – 2:40 p.m.
Conjugation of PEG (polyethylene glycol) to proteins, known
as PEGylation, has been used to improve the in-vivo lifetime
of commercial protein drugs (e.g. PEG-Intron®) in the human
body. The solution stability of such PEGylated drugs needs to be
assessed, which normally takes months. So, there is a strong desire
for alternative methods to rapidly gather information on the phase
behavior of promising target molecules. A promising alternative
suitable for miniaturization and high throughput screening is using
the osmotic second virial coefficient, B22. A correlation between
B22 and solubility and solution stability has been demonstrated.
Experimental investigation in our lab has demonstrated the use of
a chromatography based quick approach for assessing the solution
stability of PEGylated proteins through B22 screening. This
presentation will show both experimental results and theoretical
aspects of this miniaturized chromatography based quick approach
for assessing the solution stability of PEGylated proteins.
High-throughput analysis of concentration-dependent
antibody self-association
BIOT 153 – 3:40 p.m.
Shantanu V Sule, Jayapriya Jayaraman, Anna Marie Marcelino-Cruz,
Peter M Tessier, [email protected] of Chemical & Biological
Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, United
States
Monoclonal antibodies are typically monomeric and nonviscous at low concentrations, yet they display highly variable
associative and viscous behavior at elevated concentrations.
Although measurements of antibody self-association are critical
for understanding this complex behavior, traditional biophysical
methods are not capable of characterizing such concentrationdependent self-association in a high-throughput, efficient manner.
We have developed a nanoparticle-based method (self-interaction
nanoparticle spectroscopy or SINS) capable of rapidly measuring
concentration-dependent self-interactions for monoclonal
antibodies. We find that gold nanoparticles conjugated with
antibodies at low protein concentrations display self-association
behavior (as measured by the interparticle distance-dependent
Protein structure and association is important to antigenicity
in adjuvanted vaccines
James Chesko, [email protected] Disease Research Institute,
Seattle, WA 98104, United States
Recombinant proteins may act as antigens (antibody generating
molecules that induce the formation of immunoglobulins) in
biotherapeutics and vaccines. In the former case antigenicity is
an undesirable property, while in the latter it is directly linked
to the efficacy of the prophylactic. The molecular basis of how
receptors and cells and tissues of the immune system responds to
a vaccination depends typically upon biomimetic attributes of the
formulation, with the structure of the protein being a critical aspect
of the system. We will show that characteristics such as association
dramatically influence important antigens such as hemagglutinin
for influenza. Measuring such properties in modern, adjuvanted
vaccines such as emulsions can be accomplished with spectroscopic
and biophysical methods and correlated to biological activity.
69
Monday Afternoon
cells to self-renew or differentiate into specialized cell types. These
bipotent, leukemic progenitor cells differentiate into granulocytes
in response to the morphogenic compound retinoic acid (RA).
In HL60, RA treatment results in G1/G0 cell cycle arrest and
initiates production of reactive oxygen species (a function of
mature granulocytes) as the cells become committed to terminal
differentiation. Many surface proteins are upregulated in RAtreated HL60, including CD38, CD11b and BLR1. Also, sustained
activation of the Raf/MEK/ERK proteins (the MAPK signaling axis)
is a long-standing feature of RA-treated HL60. Both wild-type and
RA-resistant HL60 cells are used in concert to establish mechanistic
anomalies and identify critical differences between RA-induced
signaling in differentiation-capable and differentiation-resistant
HL60 cells.
BIOT 154 – 4:00 p.m.
Exploring Archimedes’ principle to distinguish subvisible
protein particles from silicone oil in the 0.5 um to 5 um range
Ankit R Patel, [email protected], Doris Lau, Jun Liu.Late Stage
Pharamceutical Development, Genentech, South San Francisco, California
94080, United States
While a number of characterization methods are available for
analyzing subvisible particle content in protein pharmaceuticals,
counting and characterizing particles within the entire subvisible
size range remains a challenge due to the properties of the particles
themselves and to limitations of the instrumentation. Methods
relying on differences in refractive indices of the particle and solution
(e.g. light obscuration, flow microscopy, etc.) are limited for small
particle sizes (< 2 um) while methods for characterizing smaller
aggregates (e.g. SEC, DLS, FFF, AUC, etc.) are not able to count
individual particles. Additionally, silicone oil droplets contribute to
subvisible particle counts for samples stored in prefilled syringes,
which are increasingly being used for biotherapeutic products.
Here, we evaluate a method that relies on differences in particle
buoyant mass for characterization of particles in the range of ca
0.5 – 5 um. A model particle system was specifically designed to
evaluate the suspended microchannel resonator (SMR)’s ability
to distinguish between buoyant particles (e.g. silicone oil) and
dense particles (e.g. protein particles). In addition, this emerging
technique was applied to high concentration monoclonal antibody
solutions stored in prefilled syringes in stressed stability studies. It
is shown that the SMR system can potentially distinguish between
silicone oil droplets and protein particles in a size range that is
challenging for many subvisible particle characterization methods.
BIOT 155 – 4:20 p.m.
Elucidation of acid-induced unfolding and aggregation of
human IgG1 and IgG2 Fc
Ramil F Latypov, [email protected], Sabine Hogan, Hollis Lau,
Himanshu Gadgil, Dingjiang Liu.Department of Process and Product
Development, Amgen Inc., Seattle, WA 98119, United States
Understanding the underlying mechanisms of Fc aggregation is
an important prerequisite for developing stable and efficacious
antibody-based therapeutics. In our study, high-resolution twodimensional NMR was employed to probe structural changes
in the IgG1 Fc. A series of 1H-15N HSQC NMR spectra were
collected between pH 2.5 and 4.7 to assess whether unfolding of
CH2 domains precedes that of CH3 domains. The same pH range
was subsequently screened in Fc aggregation experiments that
utilized molecules of IgG1 and IgG2 subclasses with varying levels
of CH2 glycosylation. In addition, DSC data was collected over a
pH range of 3-7 to assess changes in CH2 and CH3 thermostability.
70
As a result, compelling evidence was gathered that emphasizes the
importance of CH2 stability in determining the rate and extent
of Fc aggregation. These findings provide important insights
into the stability of Fc-based therapeutics and promote better
understanding of their acid-induced aggregation process.
BIOT 156 – 4:40 p.m.
Conformational dynamics of the N-terminal actin
binding domain of dystrophin probed using pyrene
excimer fluorescence
Surinder Singh, Krishna Mallela, [email protected].
Department of Pharmaceutical Sciences, University of Colorado Anschutz
Medical Campus, Aurora, CO 80045, United States
Mutations in dystrophin trigger muscular dystrophy, however its
structure-function relationship is less understood. Dystrophin
interacts with F-actin using its N-terminal actin binding domain
(N-ABD), which is a tandem repeat of calponin-homology (CH)
domains. Previous X-ray structure indicated that the N-ABD
is a domain-swapped dimer, with each monomer in an ‘open’
conformation in which the two CH domains do not interact.
Here we investigated the conformation of dystrophin N-ABD in
solution using pyrene excimer fluorescence. Pyrene-labeled single
cysteine mutants did not show excimer fluorescence indicating
the absence of either parallel or anti-parallel dimers. Pyrenelabeled wild-type protein showed excimer fluorescence indicating
that the two native cysteines are close in space which is only
possible if dystrophin N-ABD exists in a ‘closed’ conformation
with significant interactions between the two CH domains. Upon
binding to F-actin, excimer fluorescence increased indicating that
this domain transitions to a more compact closed conformation.
This study demonstrates for the first time the conformational
transition of a dystrophin domain during its function, and suggests
that such structural dynamics might play an important role in
controlling dystrophin function.
Tuesday Morning
Sessions
Tuesday Morning Sessions
A
B
C
8:30 a.m.
Room# 16A
Downstream Processes: Advances in Non-Chromatographic Bioseparations
A.Potty, L. Pampel, M. Siwak Papers 157-164
8:30 a.m Room# 16B
Upstream Processes: Metabolic Engineering and Synthetic Biology
K. Kao Papers 165-172
8:30 a.m. Room# 17A
Advances in Biofuels Production: Application of QbD Approaches
to Process Development
8:30 a.m.
D
E
N. McKnight, T. Cano, P. Alfonso Papers 173-180
Room# 25A Stem Cells and Tissue Engineering: Engineering of Stem Cell Expansion
and Differentiation
E. Tzanakakis, M. Kallos Papers 181-187
8:30 a.m. Room# 25B
Biophysical & Biomolecular Processes: Characterization and Protein
Engineering for Improved Biophysical Properties and Separations
D. Colby, T. Das Papers 188-195
11:30 a.m. Room# 16A
Marvin J. Johnson Award Lecture
Sang Yup Lee
71
Advances in Non-Chromatographic
Bioseparations
8:30 a.m.
Room# 16A
A.Potty, L. Pampel, M. Siwak Papers 157-164
BIOT 157 – 8:30 a.m.
Economic analysis of alternative mAb capture technologies
Alex Xenopoulos, [email protected], Ajish
traditional affinity chromatography due to its use of an expanded
bed of superparamagnetic microparticles (SPM). The efficiency of
large-scale HGMS is greatly improved if SPMs offering high binding
capacity, high magnetic susceptibility, and high magnetization are
employed. In this communication, we will describe strategies for
synthesizing SPMs that are composed of M13 viruses layer that
is assembled on a superparamagnetic core. Chemically crosslinking produced highly responsive superparamagnetic particles
with a side-on orientated, adherent virus monolayer. Alternatively,
the genetic manipulation allowed reversible assembly of the
bacteriophage on magnetic core in an end-on configuration.
Separatation of antibodies from high-protein concentration
solutions was performed in a rapid and single step. The dense SPM
core of these particles makes them highly responsive to magnetic
fields and the phage produced a high antibody binding capacity.
These new biomaterials appear to be well suited for large-scale
HGMS separation and promise to be cost effective.
Radakrishnan Potty.EMD Millipore, Bedford, MA 01730, United States
Monoclonal antibodies are currently the largest class of
biotherapeutics. Their commercial purification process is based on
a robust capture step using affinity (protein A) chromatographic
resin, guaranteeing exquisite purification and little process
development effort. The downside of protein A is its high price,
which has generated a lot of interest in alternative mAb capture
technologies, both using inexpensive resins and entirely nonchromatographic approaches (such as precipitation). We evaluated
the overall economics of those alternatives at clinical and
manufacturing scales of operation by varying resin or precipitant
price, loading capacity and number of reuses. We show that
lower resin price is not simply linked to a lower process cost, as
consumables are only one part of the overall cost structure, that
includes capital and labor costs. We discuss the trade-offs involved
in replacing a unit operation as small changes in one location can
strongly affect downstream steps in ways that are not immediately
obvious, highlighting the need for integrated process development
and modeling.
BIOT 158 – 8:50 a.m.
M13 bacteriophage-activated superparamagnetic beads for
high gradient magnetic affinity separation
Julien Muzard, [email protected], Mark Platt, Gil Lee.School
of Chemistry and Chemical Biology, University College Dublin, Dublin,
Ireland
The growth of the biopharmaceutical industry has created a
demand for new technologies for the purification of genetically
engineered proteins. High gradient magnetic separation (HGMS)
is a technology that has the potential to be increase the speed of
72
BIOT 159 – 9:10 a.m.
Clarification of recombinant proteins from high cell density
mammalian cell cultures systems using new improved depth
filters
Nripen Singh1, [email protected], KS Cheng1, Neil
Soice1, Jonathan Romero2. (1) Purification Product Development, EMD
Millipore, Bedford, MA 01720, United States (2) Biogen Idec, Cambridge,
MA 02142, United States
Increasing cell culture densities and productivities during
therapeutic protein (MAbs) production are placing a larger burden
on downstream clarification and purification operations due
to higher product and impurity levels. Controlled flocculation
and/or precipitation of mammalian cell culture suspensions by
chemical means using either acids or polymers has been used as
an alternative technology to enhance the clarification throughput
and downstream filtration operations. While flocculation is quite
effective in agglomerating cell debris and adventitious (host cell
proteins and DNA), the resulting suspension is generally not easily
separable by ordinary direct filtration methods. Traditionally,
centrifugation and a combination of filtration techniques
(tangential-flow filtration and depth filtration) have been widely
used for clarifying these pretreated cell culture broths. However,
the increased cellular debris present in these complex feed streams
can prematurely foul the membrane and commercially available
depth filters, adversely impacting their capacity and throughput. In
this work, EMD Millipore has developed novel filter designs which
when coupled with cell culture suspension optimization result in
improved primary and secondary clarification for flocculated high
cell density mammalian cell cultures systems feeds. The design
of the filter media and the interactions between the flocculation
method and the filter will be discussed. Potential benefits will be
discussed, including more robust processing without a centrifuge
and the potential to completely replace centrifugation technologies
with direct harvest filtration techniques.
BIOT 160 – 9:30 a.m.
Developing recovery clarification processes for mammalian
cell culture with high density and high solid content
Xiaoyang Zhao, [email protected], Krista Petty, Thomas McNerney,
Anne Thomas, Junfen Ma, Tim Tressel, Rob Piper.Purification Process
Development, Amgen, THousand Oaks, CA 91320, United States
Monoclonal antibody expression levels have been improved
significantly through continuous development efforts in cell line,
media and operation mode of cell culture processes. As a result,
conventional clarification technologies have been seriously
challenged in their capability to clarify feed stocks for downstream
operation. In this presentation, different flocculation techniques
will be presented that improve the clarification efficiency
and provide additional benefits in impurity reductions, thus
enabling potentially more streamlined downstream processes
without negatively affecting the product quality. Harvest process
performance with multiple flocculants was evaluated at pilot scale
using multiple monoclonal antibody molecules. The ultimate goal
is to overcome the disk-stack centrifuge solid handling limitation,
improve the depth filtration and polishing filtration performance
and eventually perform the entire harvest operation using
disposable equipment.
BIOT 161 – 10:10 a.m.
Recent advances in purification of PEGylated proteins using
charge-modified ultrafiltration membranes
Krisada Ruanjaikaen, [email protected], Andrew L
Zydney, [email protected] of Chemical Engineering, The
Pennsylvania State University, University Park, Pennsylvania 16802-4400,
United States
PEGylation, the covalent attachment of a polyethylene glycol (PEG)
chain to a small protein, has been successfully used to improve the
efficacy of therapeutic proteins. One of the challenges in producing
PEGylated proteins is the purification of the desired conjugate,
typically the singly PEGylated protein from both the residual
unreacted PEG and native protein and from multiply PEGylated
conjugates produced during the reaction. Considerable work has
focused on size exclusion and ion exchange chromatography;
however, the resolution is often poor and the overall capacity/
throughput is low due to the steric hindrance provided by the
attached PEG. The objective of this talk is to summarize recent work
on the use of ultrafiltration with electrically-charged membranes
for the purification of a model PEGylated protein.
Experimental studies were performed using α-lactalbumin
PEGylated with a 20 kDa PEG, with ultrafiltration performed using
negatively-charged composite regenerated cellulose membranes.
Data were analyzed using a recently developed theoretical
model accounting for the increase in effective size associated
with PEGylation and the ion exclusion from the PEG layer. The
PEGylated α-lactalbumin was effectively separated from the
unreacted protein and PEG using a negatively charged membrane
with relatively large pore size at low ionic strength, conditions that
maximize the electrostatic exclusion of the PEGylated protein while
allowing the unreacted (neutral) PEG and small α-lactalbumin
to pass into the filtrate. The singly PEGylated α-lactalbumin was
separated from the more highly PEGylated conjugates by exploiting
the difference in both size and net electrical charge of the different
PEGylated species; the more heavily PEGylated proteins have a
greater negative charge due to the further substitution via (positive)
lysine groups. Opportunities for the simultaneous reaction and
separation of a desired PEGylated protein are also discussed. These
results clearly demonstrate the potential of using ultrafiltration for
the production and purification of desired PEGylated proteins.
BIOT 162 10:30 a.m.
Integrating single-pass tangential flow filtration into
biopharmaceutical purification processes
Alexander Brinkmann, Joshua Souther, Matthew M Westoby,
[email protected] of Biopharmaceutical
Development, Biogen Idec, Research Triangle Park, North Carolina 27709,
United States
The concentration of product intermediates is necessary or
useful in a number of biopharmaceutical processing applications.
However, traditional fed-batch tangential flow ultrafiltration (TFF)
can be limited due to the size and minimum working volume
of TFF systems. In these situations, single-pass tangential flow
ultrafiltration (SPTFF) provides a viable solution by providing
high concentration factors without the need of a recirculation
vessel. This reduces overall system size and hold-up and enables
concentration in-line with other operations. Our team examined
the performance SPTFF in multiple bioprocessing applications
and demonstrated robustness across several products. Integration
and scale-up into existing manufacturing processes is explored and
process economics are discussed. Results illustrate the benefits of
SPTFF as a platform purification technology.
73
Tuesday Morning
Downstream Processes:
Evaluation of an alternate diafiltration strategy to mitigate
precipitation for low solubility mAbs
Eva Gefroh1, [email protected], Herbert Lutz2. (1) Purification
Process Development, Amgen Inc, Seattle, WA 98119, United States (2)
Upstream Processes:
Metabolic Engineering and Synthetic Biology
8:30 a.m Room# 16B
K. Kao Papers 165-172
EMD Millipore, Playa del Rey, CA 90293, United States
A typical monoclonal antibody (mAb) process ends with a tangential
flow filtration (TFF) step to buffer exchange and concentrate the
protein to its final conditions. The standard process begins with an
initial concentration step to a specified concentration, diafiltration
at a constant volume, followed by final concentration and product
recovery. During process development, experiments are performed
to determine the optimal protein concentration at which to perform
diafiltration – this represents the concentration at which the overall
process time and membrane area are minimized. For most mAbs,
this concentration is in the 60 to 80 g/L range. These high protein
concentrations, coupled with the moderate to high salt conditions
from the previous chromatography step, can cause low solubility
mAbs to precipitate during the TFF operation, leading to low
flux, high turbidity, and poor filterability of the final pool. In this
presentation, an alternate strategy for performing diafiltration to
circumvent the low solubility conditions will be discussed. Results
from proof-of-concept runs will be shown, along with a discussion
on control strategy and process modeling.
BIOT 164 – 11:10 a.m.
Concentration polarization based method for measuring
diffusion coefficient of macromolecules
Raja Ghosh, [email protected], Simon Lu.Chemical Engineering,
McMaster University, Hamilton, Ontario L8S 4L7, Canada
Concentration polarization of a macromolecule during
ultrafiltration is a function of its diffusion coefficient. The amount
of macromolecule accumulated due to concentration polarization
could be measured from the corresponding decrease in the bulk
feed concentration. This amount could also be correlated to the
diffusion coefficient of the macromolecule using appropriate
mathematical models. Based on this, an experimental method for
measuring diffusion coefficient of macromolecules was developed.
The method was validated using several model proteins.
74
BIOT 165 – 8:30 a.m.
Synthetic microbes engineered to fight human pathogens
LuxR). We have used directed evolution to identify a number of
EsaR variants that respond to AHL concentrations between 5 and
10,000 nM. We have also engineered the esa operon, where we have
focused on varying the location and number of EsaR binding sites.
We have characterized our new promoters using gene expression
assays and found promoters with decreased basal expression
(tighter regulation) and promoters that can be both activated
and repressed by EsaR. These new transcriptional repressors and
promoters will enable construction of novel engineered densitydependent or multicellular systems for metabolic engineering
applications.
Choon Kit Wong, Nazanin Saeidi, Mui Hua Tan, Tat-Ming Lo, Chueh Loo
BIOT 167 – 9:10 a.m.
Singapore, Singapore
Cell surveillance of quorum sensing toward reporting the
presence of contamination
Poh, Matthew Wook Chang, [email protected] of
Chemical and Biomedical Engineering, Nanyang Technological University,
Given the stalled development of new antibiotics and the
increasing emergence of multi-drug resistant pathogens, there is an
urgent need for designing a new treatment regimen for infectious
diseases. In this study, we aimed to develop a synthetic biologybased antimicrobial strategy of engineering a microbial system
that fights a human pathogen. Towards this aim, we designed
and constructed a genetic circuit that enables Escherichia coli
to sense, move, and kill a pathogenic Pseudomonas aeruginosa
strain. We demonstrated that our engineered E. coli sensed and
killed planktonic P. aeruginosa and further, inhibited the biofilm
formation. Moreover, the addition of the genetic device that
allows E. coli to move towards P. aeruginosa resulted in improved
killing efficiency. These results suggest that microbes carrying our
synthetic genetic system may provide a novel synthetic biologydriven antimicrobial strategy that could potentially be applied to
fighting P. aeruginosa and other infectious pathogens.
BIOT 166 – 8:50 am.
Repressor-based tools for cell-cell communication in
synthetic biology
Jasmine Shong, Cynthia H Collins, [email protected] of
Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy,
NY 12180, United States
A grand challenge in synthetic biology is the development of
novel intercellular signaling systems. We have engineered the acylhomoserine lactone (AHL)-dependent transcriptional repressor,
EsaR, and the esa promoter for use in synthetic microbial
communities. The availability of intercellular communicationdependent repressors will enable new circuits and network behaviors
not feasible with existing parts (e.g., the transcriptional activator
Jessica L Terrell1,2, [email protected], Hsuan-Chen Wu1,2, ChenYu Tsao2, Matthew D Servinsky3, William E Bentley1,2. (1) Fischell
Department of Bioengineering, University of Maryland, College Park,
MD 20742, United States (2) Institute for Bioscience and Biotechnology
Research, University of Maryland, College Park, MD 20742, United States
(3) Sensors and Electron Devices Division, US Army Research Laboratory,
Adelphi, MD 20783, United States
Food borne illness is a serious public health issue; once distributed,
food tainted with pathogenic bacteria can cause widespread
infection before contamination is realized and food recalled.
Current laborious methods of pathogen detection could benefit
from the sensitive molecular-recognition capabilities of cells.
The expression of bacterial virulence is primarily coordinated
by a communicative process known as quorum sensing (QS).
Accordingly, we have engineered surveyor cells that fluorescently
report the presence of autoinducer-2, a signaling molecule used for
quorum sensing. By manipulating the quorum sensing circuitry
of the surveyor cell, it monitors for signaling occurring between
bacteria, and upon recognition of autoinducer-2, the surveyor
cell then expresses a surface-displayed streptavidin binding
protein, rendering it purifiable for the retrieval and concentration
of fluorescent readout. Moreover, neighboring surveyor cells
coordinate their responses enabling an amplification of the original
signal and concentration of signal output. Furthermore, we have
engineered the sensitivity of the surveyor cells by tuning the QS
“switch” at specific thresholds of autoinducer signaling. By our
characterization data, we have demonstrated that quorum sensing,
while natively used by bacteria to coordinate multi-cellular
behavior, could in fact be harnessed as a counterstrategy for their
detection.
BIOT 168 – 9:30 a.m.
Investigation of the cRaf interactome and steady-state
multiplicity in Retinoic Acid induced differentiation of
HL-60 cells
Ryan Tasseff1, Johanna Congleton2, Andrew Yen2, Jeffrey Varner1,
[email protected]. (1) Chemical and Biomolecular Engineering, Cornell
University, United States (2) Biomedical Sciences, Cornell University,
United States
Lessons learned in model differentiation systems, such as the lineage
uncommitted human myeloblastic leukemia cell line HL-60, could
inform the analysis of more complex differentiation programs. HL60 undergoes myeloid differentiation and G1/0-arrest following
persistent MAPK activation, when exposed to All-Trans Retinoic
Acid (ATRA). The architecture responsible for ATRA-induced
persistent MAPK activation, commitment, cell-cycle arrest and
differentiation, as well as pretreatment memory effects, is poorly
understood. To this end, we measured interactions between
cRaf and a panel of possible cRaf interaction partners with and
without ATRA and the cRaf inhibitor GW5074. From this survey,
we identified a signaling circuit capable of initiating persistent
MAPK signaling following RA-exposure. We then showed that
this architecture explained both persistent MAPK activation and
ATRA memory effects in HL-60 cells. Taken together, these studies
identified a key differentiation initiation circuit whose organization
might be broadly important.
BIOT 169 – 10:10 a.m.
Metabolic engineering of CHO cells for production of
anticoagulant heparin
Leyla Gasimli1, [email protected], Jong Youn Baik4, Payel Datta1,
Bo Yang2, Xue Zhao2, Susan T. Sharfstein4, Robert J. Linhardt1,2,3.
(1) Department of Biology, Rensselaer Polytechnic Institute, Troy, NY
12180, United States (2) Department of Chemistry and Chemical
Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, United States
(3) Department of Chemical and Biological Engineering, Rensselaer
Polytechnic Institute, Troy, NY 12180, United States (4) College of
Nanoscale Science and Engineering, University at Albany – State University
of New York, Albany, NY 12203, United States
Heparin is a widely used anticoagulation medicine. Strict quality
control is required for production, even with increasing global
demand for purified product. We hypothesize that Chinese hamster
ovary (CHO) cells can be induced to produce heparin by metabolic
engineering of the native heparan sulfate (HS) biosynthetic
pathway, which shares a number of biosynthetic enzymes with
heparin. We have confirmed that CHO-S cells naturally produce all
necessary enzymes for heparin biosynthesis except N-deacetylase/
75
Tuesday Morning
BIOT 163 – 10:50 a.m.
BIOT 170 – 10:30 a.m.
Predicting immediate behaviors of engineered microbial
strains for chemical production
Joonhoon Kim1,2, Christos T Maravelias1,2, Jennifer Reed1,2, reed@
engr.wisc.edu. (1) Department of Chemical and Biological Engineering,
University of Wisconsin-Madison, Madison, WI 53706, United States (2)
Great Lakes Bioenergy Research Center, University of Wisconsin-Madison,
Madison, WI 53706, United States
Computational modeling and analysis of metabolic networks has
been successful in metabolic engineering of microbial strains for
biochemical production. We have developed a new constraintbased modeling approach (RELATCH) to predict flux distributions
in knockout mutants. We have used RELATCH to predict
flux distributions in knockout mutants and compared model
predictions to experimental datasets for E. coli, Saccharomyces
cerevisiae, and Bacillus subtilis. The results indicate that RELATCH
more accurately predicts flux distributions, as well as growth rates,
compared to existing approaches (FBA, MOMA and ROOM). We
have also developed a new bi-level mixed-integer programming
strain design approach, which uses a quadratic inner objective
function, such as MOMA or RELATCH, to identify mutant strains
with improved biochemical production. The developed approaches
extend the scope of computational strain design, and can be used
to identify novel metabolic engineering strategies for chemical
production.
76
BIOT 171 – 10:50 a.m.
Use of a hybrid genetic algorithm/flux balance analysis
approach to identify metabolic pathways not predicted by
genome annotation
for the production of biofuels, including short chain alcohols such
as ethanol, n-butanol, and isobutanol. T. thermophilus showed
high tolerance to alcohols in the medium. Taken together, these
results support the biotechnological potential of this organism for
efficient biofuels production at high temperatures.
Eddy Bautista, Joseph Zinski, Erik Johnson, Ranjan Srivastava,
[email protected] of Chemical, Materials &
Biomolecular Engineering, University of Connecticut, Storrs, CT 06269,
BIOT 172 – 11:10 a.m.
Thermus thermophilus: A model thermophilic organism for
biofuels production
Jing Lu, Aditi Swarup, K. Casey DeWoody, Maciek R Antoniewicz,
[email protected] of Chemical Engineering, University of
Delaware, Newark, DE 19716, United States
Thermus thermophilus is a thermophilic bacterium that thrives
at temperatures above 80°C. Here, we demonstrate the potential
of using T. thermophilus for the production of volatile biofuels.
T. thermophilus has a high growth rate (0.30 1/hr on minimum
medium), high biomass yield (similar to E. coli), and shows high
transformation competence making it amendable to genetic
manipulations. A current bottleneck is the limited knowledge
regarding its metabolism and growth characteristics. Here, we
reconstructed and validated the metabolic network model of T.
thermophilus HB8 using 13C tracer experiments and characterized
its metabolism using 13C-metabolic flux analysis. We performed
tolerance studies to evaluate the potential of using T. thermophilus
Critical attributes for aggregation of an Fc-fusion protein and
application for process development
Tian P Wang, [email protected], Szilan Fodor, Suminda
Hapuarachchi, Grace Jiang, Ken Chen, Izydor Apostol, Gang Huang.
Analytical Sciences, Amgen Inc., Thousand Oaks, CA 91320, United States
United States
A new strategy integrating genetic algorithms with flux balance
analysis (GAFBA) was developed to aid in fundamental studies
of metabolism, as well as to facilitate curation of genome-scale
metabolic networks. The premise for this method was based on
the observation that optimization of genome-scale metabolic
models often results in no feasible solution. It was hypothesized
that metabolic constraints were either missing or incomplete.
To determine which metabolic constraints were not fulfilled,
GAFBA evolved metabolic models by relaxing various mass
balance constraints. As a result, metabolites potentially involved in
undocumented reactions were identified.
To validate the methodology, a genome-scale metabolic model for
Mycoplasma gallisepticum was generated. After GAFBA analysis,
the model predicted an average growth rate of 0.322±0.12 h-1,
closely matching the experimentally determined growth rate
of 0.244±0.03 h-1 . To accomplish this feat, new pathways not
predicted from genome annotation were identified using the
algorithm and validated based on data in the literature.
BIOT 174 – 8:50 a.m.
Advances in Biofuels Production:
Application of QbD Approaches to Process
Development
8:30 a.m. Room# 17A
N. McKnight, T. Cano, P. Alfonso Papers 173-180
BIOT 173 – 8:30 a.m.
Post-commercial manufacturing change using QbD principles:
Development and implementation of a nanofiltration step in a
therapeutic protein process
Sundar Ramanan, [email protected] Regulatory
Affairs - CMC, Baxter BioScience, Westlake Village, CA 91320, United
States
Regulatory requirements for demonstrating product comparability
for a Post-commercial Manufacturing change are often quite
intensive, in particular for therapeutic proteins. In this talk, I
will present a case study from the approach taken during the
development and implementation of a nanofiltration unit operation
in a plasma-derived, very large therapeutic protein manufacturing
process. Using QbD principles such as prior knowledge, risk
assessment, and design-of-experiments, process design space was
developed. Comparability of process and product at bench, pilot
and manufacturing scales was also demonstrated.
Protein aggregation was observed for a purification intermediate of
an Fc-fusion protein stored at -30 °C, even though the protein was
stable at -80 °C. The protein was expressed in E. coli as inclusion
bodies, refolded and purified using multiple chromatography steps.
To study the aggregation, a series of experiments were conducted
to investigate factors contributed to the transient protein instability
during freezing. We found that the presence of free thiols in the
protein is the intrinsic cause. Partial freezing accompanied by
elevated pH and increased salt and protein concentrations were
identified as extrinsic factors contributed to the aggregation. These
results provided important insights into the improvement of the
purification process resulting in the elimination of the observed
aggregation.
BIOT 175 – 9:10 a.m.
Risk analysis of integrated process steps – a model assisted
approach
Karin Westerberg1, [email protected], Lars
Sejergaard2, Ernst Broberg Hansen2, Bernt Nilsson1. (1) Department of
Chemical Engineering, Lund University, Lund, Sweden (2) Novo Nordisk
A/S, Bagsværd, Denmark
An enzymatic modification of a therapeutic protein coupled
to size-exclusion chromatography for separation and recycling
of unreacted protein was studied. A risk based approach needs
to consider both process steps together to capture all important
interactions. This was achieved by developing mechanistic models
for the reaction and SEC steps, establishing the causal links
between process conditions and product quality. Model analysis
was used as a complement to the qualitative risk assessment, and
design space and critical process parameters were identified. The
simulation results gave an experimental plan focusing on the
“worst case regions” in terms of product quality and yield. This way
the experiments were used to verify both the suggested process
and the model results. This work shows all steps necessary for a
model assisted process analysis; from model development through
experimental verification. The methodology is well suited for a
quality by design approach to process development.
77
Tuesday Morning
N-sulfotransferase-2 (Ndst-2) and 3-O-sulfotransferase-1
(Hs3st1), which are key enzymes for anticoagulant heparin
production. Genes expressing both enzymes were stably
transfected into CHO-S cells. Clones with elevated expression
were selected for further analysis. Anti-Factor Xa assays showed
an 8-fold increase in anticoagulant activity in lysates of engineered
cells, and a 100-fold increase in culture media supernatant, when
compared with wild type CHO-S cells. LC-MS indicated increased
N-sulfonation of newly synthesized heparin. These results confirm
the activity of transfected Ndst-2 and Hs3st1. Exostose-1 and -2
were transfected into CHO-S cells to increase the amount of HS
being synthesized. Elevated production of HS was confirmed by
LC-MS. Ongoing work is directed towards balancing the activity
of transfected enzymes with endogenous enzymes in the heparin/
HS biosynthetic pathway in CHO-S cells to increase the efficiency
of HS biosynthesis. Additionally, we aim to develop assays to more
precisely monitor in vivo activity of the transfected enzymes.
BIOT 178 – 10:30 a.m.
Quality by design, biopharmaceutical manufacture
Implementation of a fully integrated QbD approach for a MAb
Kurt Brorson, [email protected] Monoclonal Antibodies,
CDER/FDA, Silve Spring, MD 20903, United States
purification process
In the biopharmaceutical industry, consistently manufacturing a
safe and high quality protein drug substance can be challenging.
Recently, a regulatory structure supporting quality by design
has been described in ICH documents Q8 through 11. Many
of the concepts espoused in these documents (e.g. risk based
aproaches, QTPP, design spaces) are universal for pharma/
biopharma manufacture and can readily be applied to bulk protein
manufacture.
BIOT 177 – 10:10 a.m.
Use of statistical modeling tools to characterize the design
space for a late-stage mammalian cell culture process
James Lambropoulos, [email protected], Lia
Tescione, Ram Paranandi, Rhiannon Quirk, Barbara Woppmann, Marty
Sinacore, Helena Yusuf-Makagiansar, Thomas Ryll.Department of Cell
Culture Development, Biogen Idec, Cambridge, Massachusetts 02142,
Karthik N Mani, [email protected] Development,
Genentech, Inc., South San Francisco, California 94080, United States
A case study of a Quality-by-Design (QbD) approach to
characterizing a Monoclonal Antibody (MAb) purification
process is presented. First, a risk ranking and filtering (RRF)
tool is used to identify parameters for process characterization.
Second, multivariate characterization studies are designed and
executed based on the output of the RRF tool. Third, the results
of the characterization studies are analyzed to evaluate the impact
of each process parameter on pre-determined Critical Quality
Attributes (CQAs). A dimensionless indicator of criticality, called
Impact Ratio, is used as a quantitative and consistent measure of
parameter impact across multiple parameters and unit operations.
Fourth, linkage studies are performed at worst-case for each CQA,
to allow a highly conservative and efficient method of exploring the
edges of the Design Space across the entire purification process. In
conclusion, the implications of process characterization results to
the MAb manufacturing Control Strategy will be discussed.
United States
We describe here the design space characterization for the
production of a recombinant human protein from a mammalian cell
culture process. These activities included (1) the establishment of a
3L scale-down model representative of the 2000 L manufacturing
process, (2) a fractional factorial parameter screening study, (3)
a response-surface modeling study, which examined pH, seed
density, harvest day, and temperature and their impact on growth,
productivity, and product quality attributes, and (4) model
verification. We found that several response parameters were well
modeled and showed a high degree of predictability, while others
had poor-fitting models. We made a number of observations based
on these individual models, including: (1) that temperature set
point was optimized for titer; (2) that decreases in pH set point or
extension of harvest day led to decreased levels of Glycan Attribute
1; and (3) that decreases in temperature or harvest day led to
increased levels of Glycan Attribute 2. The various mathematical
models that were generated from this work allowed for the
identification of process optima, the setting of action limits for the
Process Validation campaign, and the identification of critical, key,
and non-key parameters. Lessons learned from these studies will be
reviewed and recommendations made to streamline the workflow
for cell culture process characterization and optimization using
multivariate, factorial design of experiments.
78
BIOT 179 – 11:10 a.m.
Using multivariate data analysis to develop a scale-down
model for a mammalian cell culture process
Ravinder Bhatia, [email protected], Pankaj Singh, psingh4@its.
jnj.com.Department of Process Development and Manufacturing Support,
PDMS, Johnson and Johnson, Janssen R & D, Malvern, PA 19087, United
States
Multivariate data analysis was performed on data from a mammalian
cell culture process producing antibodies. Data from the small
scale and large scale bioreactor runs were used for the analysis. To
develop a scale-down model of the production bioreactor process,
data from the large scale were used as a reference dataset to set
acceptance criteria of the scale-down model. The results show that
the satellite runs conducted at small scale runs using the same
inoculum and medium lots as large scale runs were unable to
meet the acceptance criteria. Titer, viable cell density and viability
trended lower, and lactate, Na+ concentration and osmolality
trended higher in the satellite runs compared to large scale runs.
It was hypothesized that due to higher lactate accumulation in the
satellite runs more base was added to control pH at the set-point,
which resulted in higher Na+ levels and increased Osmolality
in the bioreactor. Based on the data analysis (Multivariate and
univariate), operating conditions in the 3L bioreactor runs were
adjusted to meet the acceptance criteria for a scale-down model.
In this presentation, the approach used to develop a scale-down
model using multivariate and univariate data analysis tools for
mammalian cell culture bioreactor process will be presented.
BIOT 180 – 11:10 a.m.
Stem Cells and Tissue Engineering:
Engineering of Stem Cell Expansion
and Differentiation
8:30 a.m.
Room# 25A E. Tzanakakis, M. Kallos Papers 181-187
Determination of critical process parameters based upon
impact on critical quality attributes of a mammalian cell
culture process
Patrick Laitala, [email protected] of Process
Development and Manufacturing Support, PDMS, Johnson and Johnson,
Janssen R&D, Malvern, PA 19087, United States
Upstream processes for the production of a recombinant antibody
in CHO cell culture, consisting of 5 stages including cell culture
expansion, production, clarification, direct product capture
and viral inactivation, were developed and scaled up for clinical
manufacturing. These processes are controlled by a set of defined
process parameters with proven acceptable ranges established based
on development experience. Variability in process parameters over
operating ranges may cause variability in product attributes. This
makes it necessary to determine which process parameters are
considered critical in order to establish proper control strategies
to ensure a consistent product quality profile. A criticality
assessment was completed to determine which process parameters
are considered critical based on the impact of each parameter’s
operating range on a set of product quality attributes, and based
upon the degree of uncertainty of the process information. In this
evaluation seventy process parameters were considered over the 5
stage upstream process. These process parameters were evaluated
for impact on twelve critical quality attributes that define the
antibody product. Through this evaluation a number of process
parameters were identified to be critical based upon impact on one
or more product quality attributes.
BIOT 181 – 8:30 a.m.
Image analysis method for evaluating heterogeneous growth
and differentiation of embryonic stem cell cultures
Megan M Hunt1, [email protected], Guoliang Meng2, Derrick E
Rancourt2, Ian D Gates1, Michael S Kallos1. (1) Department of Chemical
and Petroleum Engineering, Schulich School of Engineering, University
of Calgary, Calgary, Alberta T2N 1N4, Canada (2) Department of
Biochemistry and Molecular Biology, Faculty of Medicine, University of
Calgary, Calgary, Alberta T2N 1N4, Canada
Embryonic stem cells are rapidly emerging as a promising cell
source for tissue engineering applications due to their unique
characteristics – namely their ability to readily proliferate in culture
as well as their potential to differentiate into all adult cell types.
The spatial arrangement of cells and its temporal evolution can be
complex leading to heterogeneous cell density with heterogeneous
cell viability fraction. This causes spatially varying mass transfer,
fluid flow, and state of stress, which in turn leads to variations
of cell growth, viability, and differentiation. To accurately assess
cell pluripotency or lack thereof, cells are fixed and stained with
antibodies rendering them useless for further applications. A noninvasive alternative uses image analysis to determine changes in
the heterogeneity and complexity of the cell culture (Hunt et al.
2011). Here, we report on the use of image analysis techniques to
index cell growth and differentiation in human embryonic stem
cells.
BIOT 182 – 8:50 a.m.
Propagation and directed differentiation of human pluripotent
stem cells in stirred-suspension culture
Yongjia Fan1, Michael Hsiung1, Emmanuel (Manolis) S.
Tzanakakis1,2,3, [email protected]. (1) Chemical and Biological
Engineering, State University of New York at Buffalo, Buffalo, NY 14260,
United States (2) New York State Center of Excellence in Bionformatics
79
Tuesday Morning
BIOT 176 – 9:30 a.m.
States
Successful therapeutic application of human pluripotent stem cells
(hPSCs) requires their large-scale generation as stable pluripotent
cells and their differentiated functional progeny. We adopted the
stirred-suspension bioreactor platform for propagating hPSCs in a
chemically defined environment under self-renewing and directed
differentiation modes. For microcarrier culture, the low adhesion of
hPSCs on the beads is a significant bottleneck. Optimal conditions
were identified and compared to our previous methodology, the
efficiency of microcarrier seeding increased from 30% to over 50%.
The distribution of cells on microcarriers was more even resulting
in aggregate-free cultures. The concentration of cultured hPSCs
under xeno-free conditions increased 30-40 fold over 8 days and
hPSC pluripotency was ascertained by qPCR and flow cytometry.
When hPSCs were subjected to differentiation they displayed
markers characteristic of different lineages. Our studies support
the use of stirred-suspension microcarrier bioreactors for the
expansion of hPSCs under chemically defined conditions.
BIOT 183 – 9:10 a.m.
Scalable expansion of human mesenchymal stem cells using a
microcarrier-based system under serum-free and xenofree conditions
Francisco dos Santos1, [email protected], Pedro Z.
Andrade1, Manuel M. Abecasis2, Jeffrey Gimble3, Andrew Campbell4,
Shayne Boucher4, Eric Roos4, Sandra Kuligowski4, Lucas Chase5,
Mohan Vemuri4, Cláudia Lobato da Silva1, Joaquim M.S. Cabral1. (1)
Department of Bioengineering, IBB - Institute for Biotechnology and
Bioengineering, Instituto Superior Técnico, Lisboa, Portugal (2) IPOFGInstituto Português de Oncologia Francisco Gentil, Lisboa, Portugal (3)
Pennington Biomedical Research Center, Louisiana State University, Baton
Rouge, United States (4) Life Technologies, Corp., Carlsbad, California,
United States (5) Cellular Dynamics International, Madison, Wisconsin,
United States
The growing demand for clinical-scale numbers of human
mesenchymal stem cells (MSC) for cellular therapies requires a
large-scale, fully monitored and controlled culture system for MSC
production. Here we report the expansion of human MSC in a
microcarrier-based system using commercially available serumfree and xeno-free reagents (StemPro® MSC SFM XenoFree, Life
Technologies). After 14 days, BM MSC reached a maximum cell
density of 2.0x105 cells/ml (fold increase of 18), while ADSC
expanded to 1.4x105 cells/ml (fold increase of 14). Then, the
scale-up of this system was successfully achieved for BM MSC
in a 1 L fully-controlled stirred bioreactor, reaching a cell density
of 1.3±0.1x105 cells/ml (12-fold increase) after 7 days. The cells
80
maintained tri-lineage differentiation potential and retained the
MSC immunophenotypic profile.
This system can produce large numbers of high quality MSC,
representing a feasible and more efficient alternative to the
traditional cell expansion protocol for clinical-scale manufacture
of MSC.
BIOT 184 – 9:30 a.m.
Blocking Mk cell line adhesion increases polyploidization and
the potential for proplatelet formation
Alaina C. Schlinker, [email protected], David
C. Whitehead, William M. Miller.Department of Chemical and Biological
Engineering, Northwestern University, Evanston, IL 60208, United States
Megakaryocyte polyploidization, which correlates with platelet
production, is considerably lower in vitro than in vivo. When
stimulated with PMA, the CHRF megakaryoblastic cell line
immediately adheres to and spreads on tissue-culture plastic. The
cells subsequently produce proplatelet-like extensions and undergo
polyploidzation, reaching maximal mean ploidy of 5.8±0.6N by
day 11. PMA-treated CHRF cells cultured on a neutral, hydrophilic
polymer do not adhere or form proplatelet-like extensions.
Importantly, they attain much higher mean ploidy by day 11
(8.6±0.5N, P<0.005) and maintain higher viability. Subsequent
transfer to tissue-culture plastic yields faster and more extensive
proplatelet formation compared to cells initially cultured on tissueculture plastic, with a maximal increase for day-5 transfer. Initially
inhibiting and then allowing adhesion may improve the CHRF cell
model of megakaryopoiesis. Preliminary data suggest that ROCK
inhibitor and blebbistatin, which inhibit focal adhesions, also
greatly increase CHRF cell ploidy. All treatments synergized with
nicotinamide to further increase polyploidization.
BIOT 185 – 10:10 a.m.
Modulating stem cell microenvironment with microfabricated
hydrogels
Ali Khademhosseini, [email protected] of
Medicine, Brigham and Woman’s Hospital, Harvard Medical School, Center
for Biomedical Engineering, Cambridge, MA 02139, United StatesHarvardMIT Division of Health Sciences and Technology, Cambridge, MA 02139,
United States
Embryonic stem cells (ESC) are generating significant interest due
to their ability to expand in culture and differentiate into a variety of
cell types including hepatocytes, pancreatic beta cells, osteoblasts,
cardiomyocytes, endothelial cells and neural cells. These cells may
provide a potential source of cells for numerous diseases including
diabetes and liver failure. One of the main challenges preventing
ES cell based therapies reaching their full potential is the lack
methods to homogeneously direct ESC differentiation. Currently,
various microscale technologies have been adopted in the tissue
engineering field in order to specifically control the spatial and
temporal features of the cellular microenvironment. We have
recently developed a stem cell culture platform using a microwell
array. This platform demonstrated an ability to precisely control the
size and shape of the embryoid bodies (EBs). We observed that the
differentiation of EBs was regulated in a size-dependent manner.
Specifically, the larger EBs had a preference to differentiation into
cardiac cells, whereas endothelial cell differentiation was enhanced
in smaller EBs. Additionally, we combined microfabrication
techniques with photodefinable hydrogels and encapsulated cells
inside microscale hydrogel blocks generating 3D tissue architectures
in a spatially controlled manner. By using this approach, we
demonstrated a platform where individual EBs could be exposed
to different environments such as a gelatin-based matrix and a
bioinert synthetic material. We have observed that endothelial
differentiation and migration were derived only on the gelatinbased matrix side, demonstrating that microengineered hydrogels
can be used for spatially controlling stem cell behavior. In this
paper, I will present our recent results in microscale technologies
that can control the stem cell microenvironment.
BIOT 186 – 10:50 a.m.
Using a light-activated culture matrix to determine the
microenvironmental cues that initiate breast-cancer tumor
metastasis
Matthew R Chapman1, [email protected], Bo Qing2, Lydia
L Sohn2. (1) Biophysics Graduate Group, The University of California
Berkeley, Berkeley, CA 94720, United States (2) Dept. of Mechanical
Engineering, The University of California Berkeley, Berkeley, CA 94720,
United States
Many types of gels (e.g. collagen, and polyacrylamide) are currently
employed to study how cells interact with their environment. The
mechanical properties of these gels are established by the degree
of polymer crosslinking. Once synthesized, these gels have static
mechanical and chemical properties that cannot be changed. Thus,
they cannot be used to study how evolving microenvironmental
conditions affect cell behavior and signaling. Here, we describe
a light-activated culture matrix for studying how cells adapt to
dynamic microenvironmental conditions. Using a reversible,
light-mediated interaction to crosslink biocompatible polymers,
we can synthesize a 3D-culture environment whose mechanical
properties can be modulated with near-IR light. We are employing
this dynamically controllable culture matrix to investigate
how microenvironmental cues facilitate tumor progression in
breast cancer. In particular we are investigating the role of the
microenvironment in controlling the behavior (e.g. quiescence vs
proliferation) of CD24-/CD44+ tumor-initiating cells.
BIOT 187 – 11:10 a.m.
Clonal analysis of hematopoietic stem cell progeny in
microfluidic cell culture arrays
Véronique Lecault1,2,3, [email protected], William J
Bowden1,4, David JHF Knapp5, Francis Viel2,4, David G Kent5, Stefan
Wohrer5, R Keith Humphries5,6, Connie J Eaves5,7, Carl L Hansen2,4,
James M Piret1,3. (1) Michael Smith Laboratories, University of British
Columbia, Vancouver, BC V6T 1Z4, Canada (2) Centre for HighThroughput Biology, University of British Columbia, Vancouver, BC V6T
1Z4, Canada (3) Department of Chemical and Biological Engineering,
University of British Columbia, Vancouver, BC V6T 1Z3, Canada (4)
Department of Physics and Astronomy, University of British Columbia,
Vancouver, BC V6T 1Z1, Canada (5) Terry Fox Laboratory, British
Columbia Cancer Agency, Vancouver, BC V6Z 1L3, Canada (6)
Department of Medicine, University of British Columbia, Vancouver, BC
V5Z 1M9, Canada (7) Department of Medical Genetics, University of
British Columbia, Vancouver, BC V5Z 1L3, Canada
Clonal analysis of single hematopoietic stem cells proliferating
under defined conditions in vitro can provide unique insights
into their biology and regulation. We have developed a powerful
technology for such analyses by establishing a microfluidic system
that allows combined automated medium exchange, live-cell
immunostaining, and recovery of selected cells. Here we show
how this system can be used to investigate the persistence of
endothelial protein C receptor (EPCR)-positive cells derived from
purified EPCR+ CD150+ CD48- mouse hematopoietic stem cells.
These 50% longterm in vivo repopulating cells were stimulated to
proliferate under different culture conditions. The results suggest
that our microfluidic system can distinguish the clones containing
in vivo repopulating progeny of hematopoietic stem cells from the
clones lacking such cells. This technology should be useful for the
future identification of conditions that optimize hematopoietic
stem cell self-renewal divisions, a longstanding challenge for the
field.
81
Tuesday Morning
and Life Sciences, State University of New York at Buffalo, Buffalo, NY
14203, United States (3) Western New York Stem Cell Culture and Analysis
Center, State University of New York at Buffalo, Buffalo, NY 14214, United
Characterization and Protein Engineering
for Improved Biophysical Properties and
Separations
8:30 a.m. Room# 25B
D. Colby, T. Das Papers 188-195
BIOT 188 – 8:30 a.m.
Investigating the aggregation mechanism of a model,
multi-domain protein and mutational strategies to deter
non-native aggregation
Joseph A Costanzo1, [email protected], Chris O’Brien2, Erinc
Sahin2, Erin Tamargo1, Kathryn Tiller1, Anne S Robinson2, Chris J
Roberts2, Erik J Fernandez1. (1) Chemical Engineering, University
of Virginia, Charlottesville, VA 22903, United States (2) Chemical
Engineering, University of Delaware, Newark, Delaware 19716, United
States
γD crystallin is a 21 kDa, two-domain lens protein associated
with hereditary cataracts that serves as a model aggregating
system for multi-domain, β sheet-rich proteins. This system was
used to evaluate mutational strategies directed towards reducing
protein aggregation, and also to elucidate the mechanism(s)
instigating aggregation. Several point mutations were studied to
determine whether stabilizing the least stable domain, the domaindomain interface, or mutating aggregation-prone sub-sequences
best negates aggregation while maintaining conformational
stability. The protein design algorithm, RosettaDesign, was
implemented along with several aggregation calculators to predict
point mutations that would improve protein stability. These
computational tools have proven useful for identifying mutants
with increased conformational stability and reduced aggregation.
The conformational stability and aggregation kinetics of mutants
were experimentally tested against the wild type using fluorescence
spectroscopy, differential scanning calorimetry, hydrogen-exchange
mass spectrometry, chromatography, and static light scattering.
These results intend to parse the relative impact of conformational
stability versus predicted aggregation propensity for multi-domain
proteins. Additionally, the aggregation mechanism(s) of select
mutations against the wild type were compared by employing
hydrogen-deuterium exchange on aggregated species followed
by dissociation, digestion, and analysis by mass spectrometry.
Findings from these studies may identify which mutational
strategies are most effective for deterring aggregation.
82
BIOT 189 – 8:50 a.m.
BIOT 191 – 9:30 a.m.
BIOT 193 – 10:30 a.m.
Engineering aggregation-resistant, high-affinity antibodies
Biophysical analysis and antibody engineering to improve
biopharmaceutical properties of antibody therapeutics
Evaluation of utilizing high temperature stability studies
for examining excipient effects during early formulation
development of monoclonal antibodies
Joseph M Perchiacca, Moumita Bhattacharya, Ali Reza A Ladiwala,
Peter M Tessier, [email protected] & Biological Engineering,
Jirong Lu, [email protected] Discovery Reasearch, Eli
Rensselaer Polytechnic Institute, Troy, NY 12180, United States
Lilly & Co., Indianapolis, Indiana 46285, United States
High-affinity antibodies are critical for numerous detection
and therapeutic applications, yet their utility is limited by their
propensity to aggregate. Therefore, determining the sequence
and structural features that differentiate aggregation-resistant
antibodies from aggregation-prone ones is critical to improving
their activity. Our hypothesis is that the complementarity
determining regions (CDRs) – which commonly contain solventexposed hydrophobic residues to mediate high binding affinity
– contribute disproportionately to the aggregation propensity
of antibodies. Consistent with this hypothesis, we find that the
aggregation behavior of several human VH domain antibodies is
governed primarily by their CDRs, and this poor solubility can
often be localized to a single CDR loop. Moreover, we find that
charged mutations within or near hydrophobic CDRs greatly
increases antibody solubility. We will present multiple examples of
how antibodies can be engineered in a systematic manner to resist
aggregation without altering their binding affinity.
Humanized Mabs have become increasingly important therapeutic
choices in a wide array of indications. Development of stable
high concentration antibody solutions to enable subcutaneous vs
intravenous administration is very challenging due to a number
of issues such as solubility, aggregation, liquid-liquid phase
separation, cryoprecipitation, high viscosity etc. We have combined
biophysical and structural analysis to understand interactions
contributing to these issues in specific instances. Case studies will
be presented to demonstrate application of these approaches to
successfully engineer antibodies with improved properties.
BIOT 190 – 9:10 a.m.
Mitigation of monoclonal antibody viscosity by modification
of protein surface charge
Randal R. Ketchem1, Mei Han1, Logan Garrett1, Jon Woodard2, Jennifer
Litowski2, Chris Woods2, Feng He2, [email protected]. (1) Protein
Science, Amgen, Seattle, WA 98119, United States (2) Drug Product
Development, Amgen, Seattle, WA 98119, United States
High solution viscosity is a major challenge in the development
of protein therapeutics such as monoclonal antibodies. Antibodies
exhibit varying degrees of viscosity, even within a single subtype,
particularly at protein concentrations approaching 100 mg/ml
or higher. This variability is predominantly due to differences in
variable domains, and, more specifically, in the CDR regions. We
have found that in many cases the level of viscosity is related to
surface charge patch area. To test the effect of surface charge patch
area on viscosity we have selected a parental antibody with high
viscosity and have made several alanine variants. The viscosity
and pI of the variants have been measured and the sizes of the
charge patches computed. We demonstrate that reducing the size
of the surface charge patch lowers viscosity. This finding yields
information concerning one cause of viscosity and provides a
means of mitigating antibody viscosity through computationally
driven engineering.
BIOT 192 – 10:10 a.m.
Comparison of unfolding pathways of proteins in solution and
on hydrophobic surfaces as investigated by hydrogen-exchange
mass spectrometry
Adrian Gospodarek, [email protected], John O’Connell,
Erik Fernandez.Department of Chemical Engineering, University of
Virginia, Charlottesville, VA 22903, United States
It is well known that protein unfolding during hydrophobic
interaction chromatography (HIC) can lead to loss of product
activity, irreversible binding, and poor selectivity. Less certain is
the relationship between solution phase stability and how a protein
unfolds on a HIC surface. We utilize hydrogen-exchange mass
spectrometry to investigate the conformation of multiple proteins
adsorbed on HIC surfaces. Multi-domain proteins known to
unfold in solution via intermediates were adsorbed on HIC resins
of varying hydrophobicity. Exchange behavior at the domain level
is investigated with proteolytic digestion to observe at what degree
of HIC hydrophobicity individual domains unfold. We observe
that the order of domain thermodynamic stability in solution is
important in determining what order domains will unfold as HIC
surface hydrophobicity increases. Comparisons of domain free
energies of unfolding in solution and when adsorbed are made
to quantify the effect individual HIC surfaces have on domain
stability.
Hardeep S Samra1, [email protected], Brittany R Avaritt2,
Steven M Bishop1, Ambarish U Shah1, Hasige Sathish1. (1) Department
of Formulation Sciences, MedImmune, Gaithersburg, MD 20878, United
States (2) Department of Pharmaceutical Sciences, University of Maryland,
Baltimore, MD 21201, United States
Determining conditions that lead to long-term stabilization of
monoclonal antibodies is a critical step in early formulation
development. During early formulation development, various
excipients/conditions are screened for their ability to limit antibody
degradation through the use of accelerated stability studies. In this
study, multiple higher temperatures (>40°C) were investigated as
a means to accelerate the excipient screening/selection process.
Stability profiles of several monoclonal antibodies, representing
multiple IgG isotypes, were formulated in multiple classes of
excipients at various concentrations. Degradation rates obtained
at the multiple accelerated temperatures are evaluated based on
the impact of various excipient classes on stability, as well as the
role conformational stability plays at predicting high temperature
stability. The utilization of these types of high temperature stabilities,
as well as concerns and considerations for implementation of these
types of studies for use in excipient screening will be discussed.
BIOT 194 – 10:50 a.m.
Anisotropic contributions to protein-protein interactions in
salting-out and salting-in
Leigh J Quang, Stanley I Sandler, Abraham M Lenhoff, lenhoff@udel.
edu.Department of Chemical Engineering, University of Delaware, Newark,
DE 19716, United States
Protein-protein interactions play a major role in determining
the thermodynamic properties of protein solutions as well as
the phase behavior, with attractive interactions driving phase
separation. The anisotropy of shape and functionality of proteins
complicates the prediction of protein interactions, and we examine
here the possibility of predicting the conflicting trends seen in the
interactions and phase behavior of lysozyme and chymosin B; the
former shows salting-out behavior over a wide range of pH values
while the latter salts in at low salt near the pI. Our emphasis is on
identifying the orientational configurations that contribute most
strongly to the overall interactions – due to high-complementarity
interactions characteristic of molecular recognition events – and
on calculating the effect of salt on such interactions. The roles
of these high-complementarity configurations relative to other
83
Tuesday Morning
Biophysical & Biomolecular
Processes:
interactions and the ability to predict the general trend in solution
behavior (salting-out or salting-in) will be discussed.
BIOT 195 – 11:10 a.m.
Novel antibody CH2-domain based binders to nucleolin:
Isolation, characterization and improvement of stability and
aggregation
Qi Zhao1, Rui Gong1, Sandeep Kumar2, Ponraj Prabakaran1,3, Kurt
Gehlsen4, Dimiter S Dimitrov1, [email protected]. (1)
Protein Interactions Group, Center for Cancer Research Nanobiology
Program, National Cancer Institute, National Institutes of Health,
Frederick, MD 21702, United States (2) BioTherapeutics Pharmaceutical
Sciences, Pfizer Inc.,, St. Louis, MO 63017, United States (3) Basic Research
Program, Science Applications International Corporation-Frederick,
Inc.,, Frederick, MD 21702, United States (4) Research Corporation
Technologies, Inc.,, Tuscon, AZ, United States
We have developed antibody CH2-domain scaffold binders to
nucleolin (NCL) which is involved in cell proliferation, tumor
cell growth and angiogenesis. We constructed a yeast CH2-based
library and identified a clone, NCL2, which binds to NCL, and
characterized its mutants for affinity maturation. Out of 20 mutants
of NCL2, we selected 3 mutants exhibiting relatively high affinities
to NCL on yeast. However, recombinant mutants produced in E. coli
accumulated as insoluble aggregates. To find the mechanistic basis
of these aggregations, we employed computational approaches,
including homology models of all three mutants using the crystal
structure of unglycosylated CH2 domain (PDB 3DJ9) and analysis
of potential aggregation prone regions (APRs), indicated two
hydrophobic residues, V27 and L71, in which replacement of both
residues to lysine led to significantly increase of monomer ratio for
at least one clone. Further improvement of the binders to NCL is
ongoing.
Tuesday Afternoon
Sessions
Tuesday Afternoon Sessions
12:30 -2:00 p.m.
Company Lunch Seminar hosted by Fogale Biotech
“On-line biomass monitoring”
Presenter: Frank Jing
12:30 - 2:00p.m.
A
B
C
D
E
Room# 31B
Future Program/BIOT Business Meeting
Open Meeting All Are Welcome Room# 31B
2:00 p.m.
Room# 16A
Downstream Processes: Advances in Non-Chromatographic Bioseparations
A.Potty, L. Pampel, M. Siwak Papers 196-202
Upstream Processes: Metabolic Engineering and Synthetic Biology
2:00 p.m. 2:00 p.m Room# 16B
K. Kao Papers 203-210
Room# 17A
Advances in Biofuels Production: Protein Engineering for Biofuels Production
Y. Zhang, T. Liu Papers 211-217
2:00 p.m.
84
Room# Hall E
Room# 25A Stem Cells and Tissue Engineering: Engineering of Stem Cells Expansion
and Differentiation
E. Tzanakakis, M. Kallos Papers 218-225
2:00 p.m. Room# 25B
Biophysical & Biomolecular Processes: Assembly and Stability in Model
and Next – Generation Protein Systems
Z. Chen, W. Weiss Papers 226-233
6:00 p.m. -8:00 p.m. Room# Hall E
Poster Session/Reception
S. Singh, C.Collins Papers 234-321
85
Advances in Non-Chromatographic
Bioseparations
2:00 p.m.
Room# 16A
A.Potty, L. Pampel, M. Siwak Papers 196-202
BIOT 196 – 2:00 p.m.
Development of a calcium-responsive beta roll peptide as a
purification tag for non-chormatographic recombinant protein
purification
Oren Shur1, [email protected], Mark Blenner2, Scott Banta1.
(1) Department of Chemical Engineering, Columbia University, New York,
NY 10027, United States (2) Immune Disease Institute and Department of
Pathology, Harvard Medical School, Boston, MA 02115, United States
Stimulus-responsive precipitating protein tags have been explored
as a method for the non-chromatographic purification of target
proteins. In this method, a protein of interest is fused to the tag,
expressed, and subsequently exposed to some stimulus. This fusion
protein precipitates out of solution, is washed and re-dissolved
in solution. Then, if necessary, the protein of interest is cleaved
from the tag and the tag is again precipitated, leaving behind
purified protein. This approach can be very powerful for rapid
bioseparations. However, the most common existing approach
using elastic-like peptides requires a stimulus (temperature swings)
that is impractical for large scale purification. We will present our
work in the development of engineered beta roll-like peptides
which reversibility fold and precipitate in the presence of calcium
ions. We have used the system to purify maltose binding protein as
well as several other important recombinant proteins.
BIOT 197 – 2:20 p.m.
Engineering high-affinity scaffolds for protein purification
Fang Liu, [email protected], Wilfred Chen.Department of Chemical
Engineering, University of Delaware, Newark, DE 19716, United States
While protein purification has long been dominated by
standard chromatography, the relatively high cost and complex
scale-up have promoted the development of alternative nonchromatographic separation methods. Here, we reported a new
non-chromatographic affinity method for the purification of
secreted proteins. The approach is to engineer cells to secrete target
86
proteins that are fused with an affinity tag. Direct purification
and recovery can be achieved using thermo-responsive elastinlike protein (ELP) scaffolds containing different capturing
domains. Naturally occurring cohesin-dockerin pairs, which are
high-affinity protein complex responsible for the formation of
cellulosome in anaerobic bacteria, were used as the initial model.
By exploiting the highly specific interaction between the dockerin
and cohesin domain and the reversible aggregation property of
ELP, highly purified and active dockerin-tagged proteins, such
as endoglucanase CelA from C. thermocellum, chloramphenicol
acetyl transferase (CAT) and EGFP, were recovered directly from
crude cell extracts in a single purification step with yields achieving
over 90%. Incorporation of a self-cleaving intein domain enabled
rapid removal of the affinity tag from the target proteins by an
additional cycle of thermal precipitation. Because of the modular
nature of method, we will discuss the use of smaller interaction
domains for the direct purification of secreted proteins from a wide
range of recombinant hosts.
BIOT 198 – 2:40 p.m.
Purification of monoclonal antibodies by affinity precipitation
using thermally responsive elastin-like polypeptides(ELPs)
fused to IgG binding domains: High-throughput analysis and
scale up considerations
Rahul D Sheth1, [email protected], Bhawna Madan2, Wilfred
Chen2, Steven M Cramer1. (1) Department of Chemical and Biological
Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, United
States (2) Department of Chemical Engineering, University of Delaware,
Newark, DE 19716, United States
Robotic high-throughput screening was employed to investigate
the performance of an ELP-Z affinity precipitation system, and the
effects of operating conditions on IgG precipitation, elution and
product quality were examined. High levels of IgG recovery were
obtained using a range of conditions, with good results even at
low ELP:IgG molar ratios and relatively high elution pH (pH 4.2).
Product quality issues such as aggregation were observed to be very
sensitive to elution conditions and were evaluated using SEC and
CD. Conditions which exhibited both low aggregation and high
product recovery were identified using a multidimensional highthroughput screen and the robustness of the process was evaluated.
Finally, key scale-up properties such as precipitate morphology
and particle size distributions were also examined at various stages
of the process using AFM and DLS. This work sets the stage for the
industrial implementation of this technology.
BIOT 199 – 3:00 p.m.
BIOT 200 – 3:40 p.m.
Protein purification by polyelectrolyte coacervation: Influence
of protein charge anisotropy on selectivity
Practical experiences from the development of aqueous
two-phase processes for the recovery of high value biological
products
Yisheng Xu, [email protected], Paul Dubin.chemistry,
University of Massachusetts Amherst, Amherst, MA 01003, United States
The effect of polyelectrolyte binding affinity on selective
coacervation of proteins with the cationic polyelectrolyte,
poly(diallyldimethylammonium chloride), was investigated for
bovine serum albumin/b-lactoglobulin (BSA/BLG) and for the
isoforms BLG-A/BLG-B. Turbidimetric titrations were used to
define conditions of complex formation and coacervation (pHc
and pHf, respectively) as a function of ionic strength. The stronger
binding of BLG despite its higher isoelectric point is shown to
result from the negative “charge patch” on BLG, absent for BSA, as
visualized via computer modeling (DelPhi). The higher affinity of
BLG vs. BSA was also confirmed by ITC. Coacervation at I = 100
mM, pH 7, of BLG from a 1:1 (w:w) mixture with BSA was shown
by SEC to provide 90% purity of BLG with a 20-fold increase in
concentration. The relationship between protein charge anisotropy
and binding affinity, and between binding affinity and selective
coacervation, inferred from the results for BLG/BSA, was tested
using the isoforms of BLG. Substitution of glycine in BLG-B by
aspartate in BLG-A lowers pHc by 0.2, as anticipated on the basis
of DelPhi modeling The stronger binding of BLG-A, confirmed
by ITC, led to a difference in pHf that was sufficient to provide
enrichment by a factor of two for BLG-A in the coacervate formed
from “native BLG” as indicated in
Marco Rito-Palomares, [email protected], Karla Mayolo-Deloisa,
Jose Gonzalez-Valdez, Celeste Ibarra-Herrera, Mirna Gonzalez-Gonzalez,
Carolina Garcia-Salinas, Oscar Aguilar, Jorge Benavides.Centro de
Biotecnologia-FEMSA, Tecnologico de Monterrey, Monterrey, Nuevo Leon
64849, Mexico
There is an increasing need to establish selective and scalable
methods of product recovery and purification that integrate
effectively with upstream operations. The established processes
to purify biological products are characterized by a sequence
of excessive membrane and chromatography steps. It is urgent
to define bioengineering strategies to establish novel processes
that can alleviate the existing process constrains for the recovery
of biological products.The development of novel purification
processes based on two-phase extraction will be presented in this
work. In particular, the fractionation of PEGylated proteins will
be critically discussed. An attractive strategy for the recovery of
recombinant proteins from plants, particularly rhG-CSF, from
alfalfa will be examined. Finally, challenges for the establishment
of bioprocesses for the purification of stem cells will be presented.
Conclusions will be drawn concerning the strategies used to
develop the downstream processes that greatly simplify the current
way in which bioproducts can be recovered.
BIOT 201 – 4:20 p.m.
High-throughput protein purification using self-cleaving intein
and ligation independent cloning
Tiana Warren, [email protected], Michael Coolbaugh, David
Wood.Department of Chemical and Biomolecular Engineering, The Ohio
State University, Columbus, Ohio 43210, United States
We have combined Ligation Independent Cloning (LIC) with a
self-cleaving intein to create a more efficient method for protein
expression and purification. LIC is a DNA recombination method
that generates long compatible overhangs to allow the insertion
of a DNA fragment into an expression vector without the need
for target-specific restriction digests or ligation. The ∆I-CM selfcleaving intein creates an affinity tag-intein-target protein gene
fusion, where the intein cleaves the tag from the purified target
protein with a mild pH or temperature shift; thus obviating the use
of expensive proteases to release the protein from the purification
tag. Furthermore, the intein can be combined with a variety of
purification tags, including the elastin-like polypeptide tag, for
non-chromatographic purification. Through this newly developed
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Tuesday Afternoon
Downstream Processes:
BIOT 202 – 4:40 p.m.
Upstream Processes:
Metabolic Engineering and Synthetic Biology
2:00 p.m Room# 16B
K. Kao Papers 203-210
Flocculation of CHO cells for separation of recombinant
glycoproteins: Effect on glycosylation profiles
Octavio T. Ramirez, [email protected], Itzoatl A. Gomez.
Instituto de Biotecnologia, Universidad Nacional, Mexico
Centrifugation of mammalian cells is the operation most
commonly used as the first step in the recovery of recombinant
glycoproteins. However, large-scale centrifugation equipment
is expensive and undesirable cell damage can occur under this
operation. Accordingly, in this work the use of flocculants as studied
as an alternative to centrifugation for the initial clarification of two
different model recombinant glycoproteins, erythropoietin and
a monoclonal antibody, from Chinnese Hamster Ovary (CHO)
cells. Ten different polymers, including poly-L-amino acids,
polyacrylamide co-polymers and polyamides, were evaluated at
concentrations between 60 to 100 ppm. The effect on cell viability,
sedimentation rate and clarification efficiency was evaluated.
The best clarification resulted when using polyethylenimine,
whose effect on the glycosilation pattern (quality) of recovered
glycoprotein was studied. Whereas no effect on the quality and
interaction (as determined by dynamic light scattering) of the
recovered recombinant monoclonal antibody was observed,
polyethylenimine had a negative effect on the glycosylation
pattern of recombinant erythropoietin. In particular, the flocculant
removed highsialic acid glycoforms, and thus, affected the quality
of the final product. The results that will be presented will show
the cases and conditions where flocculants can successfully
replace centrifugation as the first clarification step in production
of recombinant glycoproteins and will also show the cases and
conditions were flocculation is undesirable. To our knowledge, this
is the first report of how flocculants can affect the glycosylation
profiles of recovered recombinant glycoproteins.
BIOT 203 – 2:00 p.m.
Platform pathway for synthesis of hydroxyacids as value
added products from biomass in Escherichia coli
Himanshu H. Dhamankar, [email protected], Kristala L. J.
Prather.Department of Chemical Engineering, Massachusetts Institute of
Technology, 77 Massachusetts Avenue, 66-425, Cambridge, MA 02139,
United States
3-hydroxyacids (3HA) such as 3-hydroxybutyrate and
3-hydroxyvalerate serve as monomers for the biodegradable
polyhydroxyalkanoates (PHAs) and building blocks for chiral
pharmaceuticals and chemicals. We have established a platform
pathway in E. coli that allows stereospecific synthesis of novel
straight and branched chain 3HAs using glucose and small acid
molecules as staring materials. Using this pathway we have been
successful in establishing the first biosynthetic route towards the
valuable pharmaceutical building block 3-hydroxybutyrolactone
and demonstrated synthesis of novel PHA monomers
3-hydroxyhexanoic acid and 4-methyl-3-hydroxyvaleric acid.
We have also recently uncovered a novel activity exhibited by the
thiolase enzyme employed in the pathway that may be effectively
used to enhance the array of products synthesized from this
pathway. Ongoing efforts to tap into this novel activity and to
identify alternative pathway enzymes for extending the pathway
towards additional value-added products as also enhancing these
novel activities through protein engineering will be discussed.
BIOT 204 – 2:20 p.m.
Design, engineering, and optimization of a microbial process
for manufacturing of 1,4-butanediol
Harry Yim, [email protected], Inc., San Diego, CA
goal was to develop E. coli strains to produce the industrial chemical
1,4-butanediol (BDO) from carbohydrates. BDO is a four-carbon
diol that currently is manufactured exclusively through various
petrochemical routes. Here we describe our technology platform
used to design and construct a high-performing microorganism
capable of producing BDO from glucose and sucrose. The
Biopathway Predictor algorithm, OptKnock methodology and
metabolic models were used to design the pathway and strain.
Systems biology approaches including microarrays, 13C-flux
analysis, and metabolomics were applied to characterize the
strain, identify targets for further improvement, and optimize the
fermentation process. The presentation will show our progress in
BDO titer, production rate, and yield through various strain and
process improvements, resulting in a process that is being validated
at the demo scale.
BIOT 205 – 2:40 p.m.
Elucidation of carbon-nitrogen interactions in poplar by
isotope-assisted metabolic flux analysis
Shilpa Nargund1, [email protected], Ashish Misra1, Xiaofeng
Zhang1, Gary D. Coleman2, Ganesh Sriram1. (1) Department of Chemical
and Biomolecular Engineering, University of Maryland, College Park, MD
20742, United States (2) Department of Plant Science and Landscape
Architecture, University of Maryland, College Park, MD 20742, United
States
Nitrogen crucially determines the growth and productivity of
photosynthetic organisms. However, it is often supplied by means
of artificial fertilizers, whose production is energetically intensive
and significantly relies on non-renewable fossil fuels. Therefore,
investigating nitrogen use efficiency (NUE), particularly in biofuel
crops, is a problem with wide-ranging consequences for a sustainable
economy. Toward this, we employed metabolic engineering tools
including isotope-assisted metabolic flux and network analysis to
investigate NUE and carbon-nitrogen interactions in cells of the
tree poplar (Populus trichocarpa). Poplar, a biofuel crop, uses an
array of sophisticated metabolic and regulatory mechanisms to
deposit, remobilize and thereby conserve nitrogen. The results
of our MFA experiments, in which poplar cell suspensions were
subjected to different carbon-nitrogen ratios, are anticipated to
reveal the extensive metabolic rewiring that occurs in poplar cells
when they deposit and remobilize nitrogen, and eventually provide
insights toward engineering photosynthetic organisms with high
NUE.
BIOT 206 – 3:00 p.m.
Validation of the metabolic network model for E. coli using
[U-13C]glucose and 13C-metabolic flux analysis
Robert W Leighty, Maciek R Antoniewicz, [email protected].
Department of Chemical Engineering, University of Delaware, Newark, DE
19716, United States
13C-Metabolic flux analysis is a powerful technique for measuring
intracellular metabolic fluxes in living cells. Two key assumptions
of 13C-MFA are: 1) enzymes does not discriminate between
12C-atoms and 13C-atoms; 2) the metabolic network model used
for fitting isotopomer data is accurate and complete. In this study,
we validated both assumptons experimentally for wild-type E. coli
using mixtures of natural glucose and [U-13C]glucose. E. coli cells
were grown in parallel cultures on media containing 0%, 20%, 40%,
60%, 80% and 100% [U-13C]glucose. Extracellular measurements
did not reveal any differences between the cultures. In addition,
GC-MS measurements were collected for amino acids and the
isotopomer data was fit to a large-scale metabolic network model
of E. coli. We obtained consistent flux results for all experiments.
Thus, these results provide support for both hypothesis. The
validated metabolic network model is now being used to compare
metabolism of metabolically engineered E. coli strains.
BIOT 207 – 3:40 p.m.
Engineering of Saccharomyces cerevisiae for enhanced
polyketide production
Jin Wook Choi, [email protected], Nancy A Da Silva.Chemical
Engineering and Materials Science, University of California, Irvine, Irvine,
CA 92697, United States
The yeast Saccharomyces cerevisiae is a promising host for
the synthesis of fungal polyketides and fatty acids. Using
6-methylsalicylic acid (6-MSA) as a model polyketide, we
have studied the effects of host strain attributes and precursor
availability on product synthesis. Native metabolic pathways in
S. cerevisiae were up-regulated or down-regulated to enhance
precursor production; these efforts led to up to 4-fold increases in
the 6-MSA level. Two promoter systems with induction in different
phases of growth were studied and resulted in 40-fold differences
in the 6-MSA produced. The effects of the strain and pathway
manipulations on carbon utilization were also characterized.
92121, United States
Genomatica has utilized its computational and experimental
competencies in an integrated metabolic engineering platform to
design, create, and optimize novel organisms and bioprocesses. Our
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Tuesday Afternoon
system, arbitrary protein genes can be incorporated into various
expression vectors, and their encoded proteins purified, in a nonchromatographic, high-throughput manner.
BIOT 210 – 4:40 p.m.
Construction and characterization of a sexual Escherichia coli
strain
Building electrical conduits in living cells
James D Winkler, [email protected], Katy C Kao.Department of
Heather M Jensen1,2, [email protected], Jay T Groves1, Caroline
M Ajo-Franklin2. (1) Chemistry, UC Berkeley, Berkeley, CA 94720, United
States (2) Lawrence Berkeley National Lab, Berkeley, CA 94720, United
States
Asexual microbes such as Escherichia coli lack an efficient
mechanism to exchange genetic information between individuals,
leading to competition between E. coli subpopulations with
differing beneficial mutations during evolution experiments (i.e.
clonal interference). However, high frequency recombination
strains containing chromosomally integrated fertility plasmids
are able totransfer genetic material to F- E. coliin a form of sexual
recombination. Two F plasmid gender proteins, TraS and TraT,
ordinarily prevent Hfr-Hfr conjugation, ensuring that DNA transfer
is typically unidirectional. In order to exploit this phenomenon
to create a truly sexual strain of E. coli, we have engineered two
Hfr strains to allow for efficient bidirectional (Hfr-Hfr) mating
through disruption of traST. The properties and evolutionary
dynamics of these “genderless” strains are evaluated to determine
the experimental benefit of sexual recombination.
Cellular-electrical connections have the potential to combine the
specialties of the technological world with those of the living world.
However, cell membranes are natural insulators, inherently creating
a barrier between intracellular electrons and inorganic materials.
The dissimilatory metal-reducing microbe, Shewanella oneidensis
MR-1, has overcome this barrier by using an electron transport
pathway (ETP) to transport electrons to extracellular minerals. We
seek to generalize this ability to grow electrical contacts between
microbes and inorganic materials, and thus have genetically reengineered a portion of the Shewanella ETP into Escherichia
coli. These ‘electrified’ strains exhibit ~8x and ~4x faster metal
reducing efficiency with soluble metals and insoluble metal oxides,
respectively, than wild-type E. coli. These experiments provide the
first steps towards building microbial-electrical interfaces, and
furthermore demonstrate synthetic biology’s potential to rationally
engineer cells as materials. Our next objective is to measure direct
Chemical Engineering, Texas A&M University, College Station, TX 77843,
United States
BIOT 209 – 4:20 p.m.
electrical output from the ‘electrified’ strains to an electrode.
Cloning and in vivo functional analysis of constitutive
promoter elements from Propionibacterium acidipropionici
Ehab M Ammar, [email protected], Chunxiao Zhang, Shang-Tian
Yang.The William G. Lowrie Department of Chemical and Biomolecular
Engineering, Ohio State Universtiy, Columbus, Ohio 43210, United States
Propionibacteria can be used to produce propionic acid, which
currently is mainly produced via petrochemicals. Its fermentation
production can be improved by metabolic engineering.
Unfortunately, little is known about the genetics of this bacterium.
Promoters are key elements in metabolic engineering as they
control the level of gene expression. Eleven promoters from P.
acidipropionici ATCC 4875 were cloned into a shuttle vector
with a promoterless reporter gene and transformed into the host.
Promoter strengths were analyzed as function of the reporter gene
activity. Results showed that these promoters were able to enforce
constitutive expression of the reporter gene to different levels,
both in P. acidipropionici and E. coli; however, activities in these
two hosts were not closely correlated. In addition, there were no
distinct -10 and -35 regions in these promoters. These findings
would contribute positively towards improving future metabolic
engineering of P. acidipropionici for enhanced propionic acid
fermentation.
90
Advances in Biofuels Production:
Protein Engineering for Biofuels Production
2:00 p.m. Room# 17A
Y. Zhang, T. Liu Papers 211-217
BIOT 211 – 2:00 p.m.
Cellulosome-inspired systems for improved conversion of
cellulosic biomass
Edward A. Bayer, [email protected] of Biological
Chemistry, The Weizmann Institute of Science, Rehovot, Israel 76100, Israel
Recent advances in production and application of native and
designer cellulosomes and related systems for degradation of
plant cell wall polysaccharides serve to forecast their employment
for enhanced saccarification of cellulosic biomass en route to
biofuels. Designer cellulosomes can be used as a convenient
and effective tool for assessing the comparative effects of single
enzymes and mutated derivatives within the cellulosome context.
For this purpose, a wide range of CBM-, cohesin- and dockerinbearing enzymes and/or scaffoldins can be produced at will and
allowed to self-assemble into predetermined enzyme-containing
complexes. Variations in cellulosome configuration and extension
of the designer cellulosome concept provide unique systems for
deconstruction of plant-derived polysaccharides. The talk will
summarize recent approaches and results, using permutations and
combinations of cellulosome-inspired systems.
BIOT 212 – 2:20 p.m.
Implications for catalytic mechanism and thermostability of
the novel thermophilic endo--beta-1,4-glucanase
Yan Feng, [email protected] of Life Science and
Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Endo-β-1,4-glucanase from thermophilic Fervidobacterium
nodosum Rt17-B1 (FnCel5A), a new member of glycosyl hydrolase
family 5, shows high hydrolytic activities on carboxylmethyl
cellulose, regenerated amorphous cellulose, β -d-glucan from
barley and galactomannan, with the optimum temperature of 80–
83 ◦C and the optimum pH of 5.0–5.5. Furthermore, this enzyme
is highly thermostable and has a half-life of 48 h at 80 ◦C.
To understand the structural basis for the thermostability and
catalytic mechanism, the crystal structures of FnCel5A and the
complex with glucose at atomic resolution. FnCel5A exhibited a
(β/α)8-barrel structure typical of clan GH-A of glycoside hydrolase
families with a large and deep catalytic pocket located in the
C-terminal end of the β-strands, which may permit substrate
access. A comparison of the structure of FnCel5A with related
structures from thermopile Clostridium thermocellum, mesophile
Clostridium cellulolyticum, and psychrophile Pseudoalteromonas
haloplanktis showed significant differences in intra-molecular
interactions (salt bridges and hydrogen bonds), which may account
for the difference in their thermostabilities. The substrate complex
structure in combination with a mutagenesis analysis of the
catalytic residues implicates a distinctive catalytic module Glu167His226-Glu283, which suggests that the histidine may function
as an intermediate for the electron transfer network between the
typical Glu-Glu catalytic module. These results provide substantial
insight into the unique characteristics of FnCel5A for catalysis.
To expand the capacity of FnCel5A to hydrolyze the crystalline
structures of natural cellulose, the chimeras of FnCel5A and
several carbohydrate-binding modules (CBMs) from different
microorganisms are constructed. The resulting chimeric cellulases
not only remained their endogluconase activity, but also were
suitable for hydrolyzing the crystalline cellulose. The results suggest
that the derived chimeric cellulases may become good candidates
for the efficient degradation of natural cellulose.
BIOT 213 – 2:40 p.m.
Mechanistic insight into cellulase-cellulose interaction
Jun Xi, [email protected], Sylvester Greer, Wenjian Du, Jose Rafael
Quejada, Chi Nguyen, Liming Zhao, Ahmed Bulhassan.Department of
Chemistry, Drexel University, Philadelphia, PA 19104, United States
Cellulase has been widely used to convert cellulosic biomass to
fermentable sugars for biofuel production. Currently, there is
an urgent need to develop a highly efficient cellulase to greatly
improve the efficiency of cellulosic biomass conversion. This
will require a comprehensive understanding of cellulase actions
on cellulose. Cellulase catalyzes the hydrolytic degradation of
cellulose at a liquid/solid interface. Prior to the hydrolytic cleavage,
cellulase utilizes an activity known as enzymatic decrystallization
to break up the solid aggregate of cellulose molecules to release
them from their solid aggregate into the active site of the enzyme.
We utilized nanomechanical and biophysical tools to characterize
the interaction between cellulase and both insoluble and soluble
forms of cellulose to obtain a further understanding of enzymatic
decrystallization. The mechanistic insight derived from this study
may provide new leads for designing a high-efficiency cellulase for
biofuel production.
BIOT 214 – 3:00 p.m.
Computational investigations of Trichoderma reesei Cel7A
suggest new routes for enzyme activity improvements
Gregg T Beckham1, [email protected], Christina M Payne4,
Lintao Bu1, Courtney B Taylor2, Clare McCabe2, Jhih-Wei Chu3, Michael
E Himmel4, Michael F Crowley4. (1) National Bioenergy Center, National
Renewable Energy Laboratory, Golden, CO, United States (2) Department
of Chemical Engineering, Vanderbilt University, Nashville, TN, United
States (3) Department of Chemical Engineering, UC Berkeley, Berkeley,
CA, United States (4) Biosciences Center, National Renewable Energy
Laboratory, Golden, CO, United States
The Trichoderma reesei Family 7 cellulase (Cel7A) is a key industrial
enzyme in the production of biofuels from lignocellulosic biomass.
It is a multi-modular enzyme with a Family 1 carbohydratebinding module, a flexible O-glycosylated linker, and a large
catalytic domain. We have used simulation to elucidate new
functions for the 3 sub-domains, which suggests new routes to
increase the activity of this central enzyme. These findings include
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Tuesday Afternoon
BIOT 208 – 4:00 p.m.
BIOT 215 – 3:40 p.m.
Self-immobilized cells: Concept, research progress and
applications in fuel ethanol production
Fengwu Bai, [email protected] of Life Science and
Biotechnology, Dalian University of Technology, Dalian, Liaoning 116023,
China
Some cells can flocculate spontaneously during culture and
fermentation to form flocs suitable for immobilization within
bioreactors without consumption of supporting materials.
Taking ethanol fermentation as an example, a self-flocculating
yeast was bred by the protoplast fusion technique from the
self-flocculating Schizosaccharomyces pombe and the nonflocculating Saccharomyces cerevisiae, and bioreactors suitable for
the self-immobilized yeast cells was developed, correspondingly.
Furthermore, a cascade ethanol fermentation system was optimized
and validated through a pilot plant operation, technically as well
as economically. Recently, this innovative ethanol fermentation
technology was commercialized in the BBCA fuel ethanol plant,
producing 200, 000 ton ethanol per year from starch-based
feedstocks. In addition, update research progress indicates that
stress tolerance can be improved significantly when yeast cells selfflocculate due to enhanced synergistic response, indicating that
robust yeast could be developed from the self-flocculating yeast
by engineering with the pentose metabolic pathway for ethanol
production from lignocellulosic biomass.
BIOT 216 – Withdrawn
BIOT 217 – 4:20 p.m.
Low-cost and high-energy efficiency biofuels production by
in vitro synthetic biology platform: A new biotechnology
paradigm
Y-H Percival Zhang, [email protected] Systems Engineering,
Virginia Tech, Blacksburg, VA 24061, United States
Different from living entities (e.g., microbes, plants) insisting in
wasting energy and resources for self-duplication and maintenance,
cell-free systems feature numerous advantages: easy access and
control for open systems, high product yields, fast reaction rates,
tolerance of toxic compounds, broad reaction conditions and so
on. Cell-free synthetic pathway biotransformation (SyPaB) is the
in vitro assembly of stable enzymes and (biomimetic) cofactors for
implementing complicated chemical reactions that living entities
and chemical catalysts cannot do [1-3]. In this talk, I introduce
the basic concept of SyPaB and present our efforts, for example,
high-yield hydrogen generation, enzymatic fuel cells, enzymatic
conversion of cellulose to starch, and CO2 fixation.
References
1. Zhang, Y.-H.P., J.-B. Sun, and J.-J. Zhong, Biofuel production
by in vitro synthetic pathway transformation. Curr. Opin.
Biotechnol., 2010. 21 : p. 663-669.
2. Zhang, Y.-H.P., Simpler is better: high-yield and potential
low-cost biofuels production through cell-free synthetic pathway
biotransformation (SyPaB). ACS Catal., 2011. 1 : p. 998-1009.
3. Zhang, Y.-H.P., et al., Toward low-cost biomanufacturing
through cell-free synthetic biology: bottom-up design. J. Mater.
Chem., 2011: p. DOI:10.1039/C1JM12078F.
Neural stem cells are self-renewing multipotent cells that generate
the neuronal and glial cells that exist in the mammalian central
nervous system. The expansion of neural stem cells under adherent
conditions may constitute an interesting alternative to the usual
aggregate culture, as neurospheres.
The system here described provides the high numbers of mouse
embryonic stem cell-derived neural stem cells (mESNSC) likely
required for the application of these cells in drug screening or
toxicology and is a model system for possible translation to human
cells. The culture of mESNSC under adherent conditions was
firstly optimized under static conditions, with focus on the effect
of oxygen tension. Protocols for the expansion of mESNSC on
microcarriers, in 50 mL spinner flasks were then developed and
optimized leading to an almost 35-fold increase in cell number,
after 6 days. Importantly, the expanded cells remained able to
differentiate into neuronal and glial phenotypes.
BIOT 219 – Withdrawn
BIOT 220 – 2:40 p.m.
Expansion and differentiation of human amniotic fluidderived mesenchymal stem cells in polyethylene terephthalate
based 3D bioreactors
Meimei Liu, [email protected], Shang-Tian Yang.Department of
Stem Cells and Tissue Engineering:
Engineering of Stem Cells Expansion
and Differentiation
2:00 p.m.
Room# 25A E. Tzanakakis, M. Kallos Papers 218-225
BIOT 218 – 2:00 p.m.
Design and operation of a bioreactor system for the expansion
of mouse embryonic stem cell-derived neural stem cells on
microcarriers
92
Carlos A.V. Rodrigues, [email protected], Maria M.
Diogo, Cláudia Lobato da Silva, Joaquim M.S. Cabral.Department of
Bioengineering and IBB-Institute for Biotechnology and Bioengineering,
Centre for Biological and Chemical Engineering, Instituto Superior Técnico,
Lisboa, Portugal
Chemical and Biomolecular Engineering, The Ohio State Unversity,
Columbus, OH 43210, United States
Amniotic fluid-derived mesenchymal stem cells (AF-MSCs) have
emerged as an important cell source for therapeutic transplantation.
However, little is known about AF-MSCs and their expansion and
differentiation, especially in 3-dimensional (3-D) bioreactors
for possible clinical applications. In this study, polyethylene
terephthalate (PET) was used to support the growth of AF-MSCs
in spinner flasks. The expansion rate, in vitro differentiation
capacities to adiogenic, osteogenic and chondrogenic lineages, as
well as the percentage of senescence-associated ß-galactosidase
positive cells from AF-MSCs were investigated and compared
to those of bone marrow-derived mesenchymal stem cells (BMMSCs). AF-MSCs exhibited a relatively higher proliferation
property with a much shorter doubling time than BM-MSCs. Like
BM-MSCs, AF-MSCs were successfully differentiated to adipose,
bone and cartilage cells, confirming that AF-MSCs are tripotent.
The percentage of senescent cells during subcultures of AF-MSCs
was significantly lower than that of BM-MSCs. Thus, AF-MSCs
would be a promising cell source for regenerative medicine.
BIOT 221 – 3:00 p.m.
Development of buffer supporting mesenchymal stem cell
growth in atmospheric carbon dioxide
Arthur Nathan Brodsky, [email protected], Sarah W Harcum.
Department of Bioengineering, Clemson University, Clemson, SC 29631,
United States
Stem cells provide valuable resources for both therapeutic
treatments and research in fields such as tissue engineering,
pathology, and developmental biology. As these applications
expand, improvements in stem cell expansion must be developed.
Fed-batch bioreactors allow for increased volumetric scale-up and
batch-to-batch reproducibility; however, the sodium bicarbonate
buffers utilized in current medias require carbon dioxide sparging to
maintain pH. Besides complicating system controls, carbon dioxide
sparging can reduce oxygen supplies, which in turn can prevent
higher cell densities from being obtained. This study developed
a buffer that de-emphasizes sodium bicarbonate’s contributions,
which eliminates the need for carbon dioxide sparging. Without
any adaptation period, murine mesenchymal stem cells cultured
with HEPES-MOPS buffer in atmospheric carbon dioxide grew at
rates equal to those achieved in high carbon dioxide environments.
Retention of multipotency and differentiation patterns for stem
cells, and application to suspension cultures will be discussed.
BIOT 222 – 3:40 p.m.
Effect of gold nanoparticles on adipose derived stromal cells
Tatsiana Mironava1, [email protected], Michael
Hadjiargyrou2, Marcia Simon3, Miriam H Rafailovich1. (1) Department
of Materials Science, Stony Brook University, Stony Brook, NY 11794,
United States (2) Department of Biomedical Engineering, Stony Brook
University, Stony Brook, NY 11794, United States (3) Department of Oral
Biology and Pathology, School of Dental Medicine, Stony Brook University,
Stony Brook, NY 11794, United States
Gold nanoparticles (AuNPs) are being used for diagnostic and
therapeutic purposes; however, their potential health risk(s)
are still under investigation. Various reports have shown that
nanobased materials can facilitate stem cell therapy for bone tissue
engineering and wound healing.
In this study we have investigated the effect of AuNPs on human
adipose derived stromal cells (ADSc) and found that the uptake
of the AuNPs is a function of time, their size and concentration
as well as culture media used. Exposure to AuNPs affects the
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Tuesday Afternoon
new roles for glycosylation, which we have shown can be used to
tune the binding affinity. We have also examined the structures of
the catalytically-active complex of Cel7A and its non-processive
counterpart, Cel7B, engaged on cellulose, which suggests allosteric
mechanisms involved in chain binding when these cellulases are
complexed on cellulose. Our computational results also suggest that
product inhibition varies significantly between Cel7A and Cel7B,
and we offer a molecular-level explanation for this observation.
Finally, we discuss simulations of the absolute and relative binding
free energy of cellulose ligands and various mutations along the
CD tunnel, which will affect processivity and the ability of Cel7A
(and related enzymes) to digest cellulose. These results highlight
new considerations in protein engineering for processive and nonprocessive cellulases for production of lignocellulosic biofuels.
BIOT 223 – 4:00 p.m.
Engineering cell-material interfaces for long-term expansion
of human pluripotent stem cells
Yongsung Hwang1, [email protected], Chien-Wen Chang2,
David Brafman3, Thomas Hagan1, Shyni Varghese1. (1) Department of
Bioengineering, University of California, San Diego, La Jolla, CA 920930412, United States (2) Department of Biomedical Engineering and
Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan
Republic of China (3) Department of Cellular and Molecular Medicine,
University of California, San Diego, La Jolla, CA 92093-0695, United States
Developing cost-effective and scalable synthetic matrices for
long-term expansion of human pluripotent stem cells (hPSCs)
is important to generate large number of cells required for their
clinical application. Here, we discuss the development of a synthetic
hydrogel containing heparin-mimetic moieties to support longterm expansion of hPSCs for over 20 passages. HPSCs expanded
on this synthetic matrix maintained their characteristic colony
morphology, karyotypic stability, and pluripotency. Additionally,
we identify correlations between various material properties,
such as functional group, hydrophobicity, charge density, and
rigidity, on various cellular responses leading to control the fate
and commitment of hPSCs. The observed cellular responses are
explained through matrix mediated binding of ECM proteins
and growth factors. Such synthetic matrices comprising of “offthe shelf ” components are easy to synthesize and do not require
any sophisticated processing thus making them cost-effective and
translational.
assembly peptide scaffold and cultured in a two-chamber well for
one step osteochondral multilayered constructs generation in vitro
was investigated. This co-culture system could provide osteogenic
and chondrogenic stimulations to BMSCs concurrently on the
different layers of scaffold. Results demonstrated that this co-culture
approach could successfully provided zones differently chemical
stimulations to BMSCs on different layers in a single scaffold, which
resulted the formation of the multilayered osteochondral constructs
with cartilage like, osteochondral interface and subchondral bone
like tissues. Cells at intermediated region appeared hypertrophic
chondrocytes morphology. These cells embed in calcified ECM
which was also rich in glycosaminoglycans, type collagen I, II and
X. In conclusion, this study provided a one-step approach that
choosing BMSCs as only cell sources seeded on single scaffold for
osteochondral multilayered constructs regeneration.
viability is retained on the chip platform. Additionally, we utilized
a chip-based in situ immunofluorescence assay that provides
quantitative information on cellular levels of proteins involved in
neural fate. We are thus able to study expansion and differentiation
of NSCs upon removal of growth factors, by monitoring the levels
of several neural progenitor and differentiation markers. The
versatility of the platform was further expanded by complementing
the cell culture chip with a chamber system that allowed us to
screen for differential toxicity of small molecules to hNSCs.
BIOT 225 – 4:40 p.m.
Assembly and Stability in Model and
Biophysical & Biomolecular
Processes:
Kelei Chen, [email protected], Siew Lok Toh, Cho Hong, James
Goh.Department of Bioengineering, National University of Singapore,
Singapore, Singapore
The regeneration of complete osteochondral constructs with a
physiological structure is a significant issue. In this study, a method
that used bone marrow stromal cells(BMSCs) with silk/self-
94
BIOT 227 – 2:20 p.m.
3Dl cellular microarray platform for human neural stem cell
differentiation and toxicology studies
Molecular simulations of polymorphic human islet amyloid
polypeptide (hIAPP) oligomers
Luciana Meli1, [email protected], Helder S. C. Barbosa1,2, Maria
Jun Zhao, [email protected], Xiang Yu, Qiuming Wang, Chao Zhao,
Margarida Diogo2, Joaquim M.S. Cabral2, Robert J. Linhardt1, Jonathan
S. Dordick1. (1) Department of Chemical and Biological Engineering,
Rensselaer Polytechnic Institute, Troy, New York 12180, United States (2)
Center for Biological and Chemical Engineering, Instituto Superior Técnico,
Jie Zheng.Department of Chemical and Biomolecular Engineering, The
University of Akron, Akron, OH 44325, United States
Lisboa, Portugal, Portugal
We developed a three-dimensional (3D) cellular microarray
platform for the high-throughput analysis of human neural stem
cell (hNCS) differentiation and toxicity screening. The growth
of an immortalized hNSC line, ReNcell VM, was evaluated on
a miniaturized cell culture chip consisting of 30 nl spots of cells
encapsulated in alginate, and compared to standard culture
conditions. Using a live/dead assay, we demonstrated that cell
Next – Generation Protein Systems
2:00 p.m. Room# 25B
Z. Chen, W. Weiss Papers 226-233
BIOT 224 – 4:20 p.m.
Generation of osteochondral multilayered constructs by bone
marrow stromal cells in vitro
A hallmark of Alzheimer’s disease is deposition of insoluble
amyloid-beta (Abeta) fibrils in the brain. Due to unusually high
stability of amyloid fibrils with cross-stacked beta-sheet structure,
conversion of oligomers/protofibrils to fibrils is often considered
as an irreversible process. Recently, we discovered that two
biocompatible, blood-brain barrier permeable molecules (brilliant
blue G and erythrosine B) can modulate Abeta aggregation and
cytotoxicity. Combined with another Abeta aggregation modulator
(methylene blue), we explored whether these molecules can reverse
the fibril formation process. Our preliminary results indicate
that all three molecules lead to substantial secondary structure
change and/or disintegration of Abeta40 fibrils. We plan to explore
thermodynamic features of amyloid fibrils and small-molecule
induced conformers and determine factors critical to reverse the
amyloid fibrillization process.
BIOT 226 – 2:00 p.m.
Misfolding and self-assembly of human islet amyloid polypeptide
(hIAPP) into polymorphic amyloid oligomers is pathologically
linked to type II diabetes. But, atomic structure and biological role
of these hIAPP oligomers are still unclear. Here, a computational
framework is developed to search for a diverse set of hIAPP
oligomers with the lowest energy landscape, two “stackingsandwich model” and “wrapping-cord model” are proposed to
describe polymorphic structures of hIAPP oligomers, and all-atom
molecular dynamics simulations are used to examine the structure,
dynamics, and interactions of the self-assembled hIAPP oligomers.
Seven oligomers from the stacking-sandwich model and three
oligomers from the wrapping-cord model are determined by
their high structural stability with favorable peptide-peptide
interactions, although all of them display completely different
structures in symmetry and β-sheet packing. These oligomeric
structures can also serve as templates to present double- and
triple-stranded helical fibrils via peptide elongation, explaining the
polymorphism of amyloid oligomers and fibrils.
Secondary structure changes and disintegration of amyloidbeta fibrils mediated by biocompatible, blood-brain barrier
permeable small molecules
BIOT 228 – 2:40 p.m.
Inchan Kwon, [email protected], Jacob A Irwin.Department of
Gaius A Takor1, Seiichiro Higashiya1, Mirco Sorci2, Natalya I Topolina3,
Chemical Engineering, University of Virginia, Charlottesville, Virginia
22904, United States
Chimera-induced folding: Implications for amyloidosis
John T Welch1, [email protected], Georges Belfort2. (1) Department
of Chemistry, University at Albany, Albany, New York 12222, United
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Tuesday Afternoon
ADSc differentiation and results in the adipogenesis suppression
by down regulation of adiponectin, known adipogenesis marker,
and lipid accumulation inhibition. ADSc fibronectin expression
was also inhibited by AuNPs as well as cell migration and collagen
contraction.
Lastly, our data indicates that AuNPs damage to ADSc is not
permanent and that cells recover as a function of lipid accumulation.
Non-native proteins influence amyloidosis and multiple protein
misfolding diseases suggesting that cross-seeding may be critical
to promoting misfolding. A synthetic chimeric amyloidogenic
protein (YEHK21-YE8) composed of two components – one
that readily folds to form fibrils (YEHK21) and one that does
not (YE8)- was prepared to facilitate study of induced folding.
Secondary structural conformational changes during aggregation
of YEHK21-YE8, demonstrate that YEHK21 is able to induce fibril
formation of YE8 unambiguously demonstrating induction of
folding and fibrillation within a single molecule.
Aggregation remains a leading problem in the development of
stable biological therapeutics and is a basic barrier to peptide and
protein-based drugs. There is a tremendous need to understand
how this process is modulated by amino acid sequence and how
it responds to cues spanning temperature, pH, salt, solvent, and
BIOT 229 – 3:00 p.m.
Identification of peptide sequences to form amyloid-like fibrils
BIOT 232 – 4:20 p.m.
Qiuming Wang, [email protected], Xiang Yu, Jun Zhao, Chao
Zhao, Jie Zheng.Department of Chemical and Biomolecular Engineering,
the University of Akron, Akron, Ohio 44325-3906, United States
Identification of the fibril-forming peptide sequences is biologically
important for understanding the sequence-structure-activity
relationship of amyloid diseases and for finding potential inhibitors
and therapeutic strategies against amyloid diseases. Here, we
marry QSAR model, molecular dynamics (MD) simulations, and
biophysical experiments to search for hexapeptide sequences with
high tendency to form amyloid-like fibrils. We first use existing
amyloid sequences to develop a QSAR model which predicts
amyloid-forming sequences from a complete hexapeptide library.
The preselected sequences from the QSAR model are examined
their ability whether or not to form β-sheet structures using
MD simulations. Ten computationally screened and designed
sequences are further experimentally validated for their ability to
form β-sheet-rich fibrils using AFM, CD, FTIR, and X-ray. These
self-assembly hexapeptides could serve as potential inhibitors to
interfere with naturally-occurring amyloid peptide self-assembly
and thus to prevent amyloid fibril formation.
BIOT 230 – 3:40 p.m.
Understanding peptide oligomerization and aggregation with
a multiscale simulation approach
M. Scott Shell, [email protected], Scott Carmichael, Joohyun
Jeon.Department of Chemical Engineering, University of California - Santa
Barbara, Santa Barbara, CA 93106, United States
96
by several factors such as temperature and protein sequence.
Mutation of one amino acid in a protein can dramatically affect
the aggregation propensity. Here we analyze, via coarse-grained
molecular modeling, the role of these interactions on the stability
and aggregation propensity of human γD-Crystallin, a natively
monomeric two-domain protein. By considering proteins as folded
rigid bodies that interact through van der Waals, hydrophobic, and
screened Coulomb forces between individual amino acids, the
thermodynamic stability of the wild type protein, as well as a series
of surface charge mutants, is evaluated. RosettaDesign is also used
to minimize disruption of conformational stability as part of the
design strategy. Interestingly, no simple relation between PPI and
aggregation propensity was found, as, for instance, changes on the
protein net charge do not always yield the same effect on inherent
aggregation propensity –quantitatively or qualitatively. The results
are compared against experimental data for some of these mutants,
suggesting the methods may be applicable more generally.
Laurence1. (1) Department of Pharmaceutical Chemistry, University of
Kansas, Lawrence, KS 66047, United States (2) Department of Chemistry,
University of Kansas, Lawrence, KS 66045, United States
Targeted delivery of cytotoxins represents the frontier of cancer
therapy in which antibody-drug conjugates (ADCs) are utilized
to deliver organic toxins. Platinum compounds are the most
commonly utilized cytotoxins, but a viable conjugate system has
not yet been developed. We generated an approach for targeted
delivery of platinum to cancer cells by including a tripeptide
carrier inline with a targeting protein and inserting metal via a
novel reaction. Platinum binding to our carrier is irreversible at
physiological pH; the complex is extraordinarily stable, but the
metal is released upon acidification, permitting controlled release.
Our unique system provides the ability to rationally design and
site-specifically encode the tripeptide into desired positions
within a protein, generating a homogeneous product, which can
be well-characterized. Metal-containing conjugates pose different
challenges for assessment of the protein product and its overall
stability. Here we describe development and characterization of
our metal-carrier system.
Amyloid ion channels: Selective or non-selective channel?
chemical additives. Here, we use new molecular simulation
methods to understand the origins or sequence-specific aggregation
behavior. Using all-atom simulations, we show that computed free
energies of small oligomer formation recapitulate experimental
fibril formation propensities for a family of peptides and suggest
an unexpected new entropic stabilization force that results from
a coupling of hydrophobic and electrostatic interactions. We also
use a novel coarse-graining theory to develop simpler molecular
models from these results that can then be deployed in larger-scale
simulations. The coarse-grained models created in this way capture
beta-sheet formation and suggest mechanisms of fibril formation.
BIOT 231 – 4:00 p.m.
Applying coarse-grained molecular models for rational
design of colloidal protein-protein interactions to mitigate
aggregation
Marco A. Blanco1, Christopher J. O’Brien1, Joseph A. Costanzo2, Erik
J. Fernandez2, Anne S. Robinson1, Christopher J. Roberts1,
[email protected]. (1) Department of Chemical Engineering, University of
Delaware, Newark, DE 19716, United States (2) Department of Chemical
Engineering, University of Virginia, Charlottesville, VA 22904, United
States
Colloidal protein-protein interactions (PPI) in solution have a
dramatic effect on protein stability and protein self-assembly. These
interactions are important for applications ranging from protein
phase behavior to aggregation of biopharmaceuticals. However, the
nature of these interactions is poorly understood since it is affected
Jie Zheng1, [email protected], Jun Zhao1, Xiang Yu1, Hyunbum
Jang2, Ruth Nussinov2. (1) Department of Chemical and Biomolecular
Engineering, University of Akron, OH 44325-3906, United States (2)
Center for Cancer Research Nanobiology Program, SAIC-Frederick, Inc.,
National Cancer Institute-Frederick, Fredrick, MD 21702, United States
The toxicity of amyloid peptides is strongly associated with their
interactions with cell membranes. The formation of ion-channel by
inserting amyloid peptides in cell membranes is proposed to enable
the unregulated passage of ions cross the membrane and to induce
cellular ionic homeostasis. But, it is still unclear whether or not
these amyloid ion channels can induce selective ion permeability.
Here, we model a series of Aß and hIAPP ion channels in lipid
bilayers to examine the effects of peptide sequences, sizes and
structures of ion channels, and lipid compositions on the ion
permeability and specificity. It is interesting to observe from
molecular dynamics simulations that Aß ion channels displays high
cation electivity especially for Ca2+ over other ions, while hIAPP
ion channels exhibit poor cation selectivity, strongly depending on
channel structures. A postulated mechanism is proposed to explain
the difference between selective Aß and non-selective hIAPP ion
channels.
BIOT 233 – 4:40 p.m.
Characterization of platinum-protein conjugates for targeted
anti-cancer therapy
Mary E. Krause1, [email protected], Brittney J. Mills2, Jennifer S.
Poster Session/Reception
6:00 p.m. -8:00 p.m. Room# Hall E
S. Singh, C.Collins Papers 234-321
BIOT 234
Expression and purification of EsaI AHL synthase enzyme
Miranda Ghali1,2, [email protected], Rajesh Nagarajan1,
Jesse Keeler1, Remington Turner1. (1) Department of Chemistry and
Biochemistry, Boise State University, Boise, Idaho 83725, United States (2)
Department of Chemistry and Biochemistry, Stetson University, DeLand,
FL 32723, United States
Bacterial infections have become more difficult to treat as
communication between bacteria allows for growth and resistance.
Bacteria use autoinducer molecules to take a census count of
neighboring bacteria in the colony. The autoinducer molecules
are synthesized inside the cell by a dedicated set of enzymes
and are released outside to aid in intercellular communication.
This mechanism of communication, known as quorum sensing,
allows bacteria to coordinate their activities that include secretion
of virulence factors and formation of biofilms. EsaI is an AHL
Synthase enzyme in the plant pathogen Pantoea stewartii, which
97
Tuesday Afternoon
States (2) Department of Chemical and Biological Engineering, Rensselaer
Polytechnic Institute, Troy, New York 12180, United States (3) Department
of Biological Sciences, University of Albany, Albany, New York 12222,
United States
BIOT 235
Filamentous freshwater biomass as a bioenergy source
Nancy L Paiva, [email protected], Stefan T. Jones, Tetchi
H. Assamoi, Judith Zounon, Steve McKim.Department of Chemistry,
Computer & Physical Sciences, Southeastern Oklahoma State University,
Durant, OK 74701, United States
A potential biomass resource for alternative fuels production is
the floating filamentous freshwater algae and plants common in
rural Oklahoma. In preliminary studies, large samples of algae and
duckweed (Lemna sp.) were collected by simply skimming pond
surfaces. Samples were sun-dried and powdered. Energy contents
of algal biomass samples were measured through oxygen bomb
calorimetry and compared to switchgrass, a land-based Oklahoma
biomass crop. Total C, N and mineral content were determined.
Algae samples from two harvests had slightly lower energy
contents (14.4+0.3 & 15.5+0.95kJ/g) than switchgrass samples
(17.0+1.0kJ/g). Algae samples had similarly lower total carbon
content (38%C) compared to switchgrass (44%C). Dried algae
contained 50 to 100 times higher Fe and Mn levels compared to
switchgrass. Analysis of duckweed biomass is ongoing. Currently,
slow pyrolysis of dried aquatic biomass is being explored to produce
crude bio-oil, for conversion to stable fuels. (Funding: OklahomaNSF-EPSCoR #EPS-0814361 and NASA Oklahoma Space Grant.)
BIOT 236
Efficient treatment of diazinon pesticide using
organophosphorus hydrolase through ultrasonic celllysis process
Suk Soon Choi1, [email protected], Sang Hwan Seo1, Hyun
Min Lee1, Chang Sup Kim2, Hyung Joon Cha2, Inchan Kown Kwon3.
(1) Department of Biological and Environmental Engineering, Semyung
University, Jecheon City /Shinwol Dong, Chungbuk 390-711, Republic of
Korea (2) Department of Chemical Engineering, Pohang University of
Science and Technology, Pohang City/Hyoja Dong, Kyungbuk 790-784,
Republic of Korea (3) Department of Chemical Engineering, University of
Virginia, Charlottesville, Virginia VA 22904, United States
In the present work, diazinon pesticide which is known as
nondegradable and neurotoxic material was removed by
organophosphorus hydrolase (OPH). 25℃ culture temperature and
98
addition of 0.2 mM ethylenediamine tetraacetate were optimal
conditions for the OPH production in Escherichia coli. 25 and 50
mg/L diazinon were treated efficiently with all over 90% removal
efficiencies using cell lysates through ultrasonication process.
Thus, a novel method developed in this work could be applied to
the bioremediation technology in the contaminated region with
high diazinon concentration.
BIOT 237
strategies compared to traditional larger scale vessels. A variety
of engineering aspects of a small scale bioreactor will be explored
which will include pH, temperature and dissolved oxygen control
under multiple operating conditions (volume, mixing, and vessel
configuration). Data will also be shown where a high-throughput
bioreactor, Micro-24 Microreactor was used as part of the
selection process of a “winning” clone/process that uses Pfenex’s
Pseudomonas fluorescens-expression system, with a discussion of
the subsequent scale up of the clone and process.
Photosystem I based solar cell for on-site hydrogen production
BIOT 239
David R Baker, [email protected], Amy K Manocchi, Scott
Pendley, James J Sumner, Margaret Hurley, Kang Xu, Barry D Bruce,
Cynthia A Lundgren.U.S. Army Research Laboratory, Adelphi, MD 20783,
Investigation of metabolic changes upon hyperosmotic stress in
monoclonal antibody producing Chinese hamster ovary clones
United States
Laura Morris, [email protected] of Nanoscale Science and
Photosystem I (PSI), a key protein in the photosynthetic pathway,
is an ideal structure for photocatalyically producing hydrogen gas
because of its high quantum yield and long-lived excited state. To
date, PSI has generated hydrogen in suspensions from H+ ions
with the aid of sacrificial electron donors and bound platinum
nanoparticles. PSI is here electrophoretically deposited onto
an electrode producing a solar cell with high densities of PSI in
electrical contact with the substrate and is able to demonstrate
electrochemical activity. By concentrating onto an electrode, the
PSI no longer needs an electron donor eliminating the need for
logistically hazardous fuels, and is able to become a more portable
technology useful for splitting grey and wastewater in the field. The
project is approached as a collaborative effort employing multiscale
modeling techniques and experimental approaches to efficiently
develop a functional and optimized system.
BIOT 238
Characterization of high-throughput 10 mL bioreactors for cell
line selection and process development
Tiffany D Rau1, [email protected], Torben R Bruck2, tbruck@
pfenex.com, David Loer2, Lacey Douthit2, Savanah Howe2, Lawrence
Chew2. (1) Pall Corporation, Port Washington, NY 11050, United States
(2) Pfenex Inc, San Diego, California 92121, United States
Cell lines and processes not only need to be developed rapidly to
meet commercial timelines, but cell based processes need to be
robust so that may be easily transferred from small scale (R and
D) to commercial scale (Design for Manufacturing). Small scale
high-throughput bioreactors allow for cell lines to be screened
controlled in both the microbial and mammalian world rapidly
and earlier than ever before with fewer resources. Typically small
scale devices often use different sensor technologies and mixing
Engineering, United States
Monoclonal antibodies are homogenous immunological
reagents of defined specify that are gaining importance as
biotherapeutics. We have concluded from previous research that
if we induce hyperosmotic stress in CHO cell cultures by adding
osmolytes (specifically sodium chloride) there is an increase in
specific antibody productivity. However, there is not an overall
significant yield increase due to the decrease in cell viability and
cellgrowth. The objective of this research is to better understand
the molecular mechanisms responsible for the uncharacteristic
globular metabolic changes that genes experience under osmotic
stress and to determine genes that have an increase in productivity
during stress but do not go undergo apoptosis. DNA microarrays
were used to investigate transcriptional changes in both CHO
and murine hybridoma cells (which also produce monoclonal
antibodies) in order to reveal the mechanisms responsible
for increasing specific antibody productivity and reduced cell
growth. However transcriptomic studies return a large number of
differentially expressed genes, many of which will not play a role
in the phenotypes of interest. To identify genes of interest, the
Sharfstein group has developed novel computational approaches
to analyzing microarray results, which have been employed to
identify putative genes of interest (including genes that were not
observed to be differentially expressed in the microarray screens).
The objective of this work is to validate the computational results by
performing quantitative real time PCR on RNA from other CHO
cell lines that produce recombinant monoclonal antibodies.Protein
quantification will be conducted through the process of Western
blotting against the protein of choice. We believe that there is a
strong correlation between the productivity and the expression of
certain genes.
BIOT 240
Adhesion and proliferation of the microalgae Chlorella
vulgaris on polymer surfaces
Min Hao Wong1, [email protected], Chunlin He2, Julie Stiver2,
Mark P. Stoykovich2. (1) Department of Chemical and Biomolecular
Engineering, University of Illinois, Urbana Champaign, UrbanaChampaigh, IL 61801, United States (2) Department of Chemical and
Biomolecular Engineering, University of Colorado at Boulder, Boulder, CO
80309, United States
The adhesion and growth behavior of cells supported on substrates
can be significantly influenced by surface properties, including the
surface charge, functional group chemistry and roughness. Surfaces
that exhibit low toxicity, good sorption capacity, chemical stability
under experimental conditions and, optionally, high selectivity are
often referred to as “growth substrates”. Some growth substrates
have been found to affect the adhesion, growth, morphology and
differentiation of various cell types. The response of cells to a given
surface, however, is often cell type specific. Here we characterize
the effect of a range of charged and uncharged polymeric surfaces
on the adhesion and growth behavior of the green microalgae
Chlorella vulgaris, a prototypical freshwater species being studied
for algal biofuels applications. Results suggest that surface
characteristics such as charge properties and functional groups
modulate the adhesion of C. vulgaris. Possible physiochemical and
biological relationships that govern the behavior of C. vulgaris on
surfaces were also explored.
BIOT 241
Enzyme engineering of malonyl coA reductase (MCR) to
improve 3-hydroxypropionate (3HP) production
Faith D Watson, [email protected], Christopher P Mercogliano,
Wendy K Ribble, Rebecca D Anderson, Hans H Liao, Tanya W Lipscomb,
Michael D Lynch.OPXBIO, Boulder, CO 80301, United States
3-hydroxypropionate (3HP) can serve as a key intermediate in
the production of several commodity chemicals. One enzyme
able to produce 3HP is the malonyl-CoA reductase (MCR)
from Chloroflexus aurantiacus. This enzyme performs two
NADPH-dependent steps to reduce malonylCoA to 3HP. This bifunctional enzyme consists of an N-terminal short-chain alcohol
dehydrogenase domain and a C-terminal aldehyde dehydrogenase
domain. By constructing truncated versions of this enzyme by
removing ~150 amino acid increments from the C-terminal
and N-terminal region of the protein, we were able to define
the boundaries of these individual domains. Once determined,
we performed a ‘cofactor switching’ procedure on each of these
domains to redesign MCR’s co-factor preference from NADPH
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Tuesday Afternoon
uses S-adenosyl-L-methionine and 3-oxohexanoyl-ACP to make
3-oxohexanoyl-HSL autoinducer molecules inside the cell. We
expressed EsaI AHL Synthase as a his-tagged protein and purified it
using Ni-NTA chromatography. The purity of EsaI was confirmed
by SDS-PAGE gel electrophoresis. EsaI enzyme will be subjected to
mechanistic studies in the future.
BIOT 244
BIOT 242
Satoshi Katahira1, [email protected], Kenro Tokuhiro1,
Application of RT-PCR analysis of pig muscle tissue mRNA
expression for ChREBP gene sequence
Edward J Parish1, Xiao Meng2, Hiroshi Honda2, chrishonda@yahoo.
com. (1) Department of Chemistry and Biochemistry, Auburn University,
Auburn, AL 36849, United States (2) Department of Bioengineering,
Northwestern Polytechnic University, Fremont, CA 94539, United States
The carbohydrate response element binding protein, ChREBP, can
activate mutiple glycolysis and fat synthesis, gene transcription,
thereby regulating glucose metabolism and fatty acid synthesis.
This paper represents using RT-PCR technology to obtain ChREBP
gene for gene sequence.
BIOT 243
Regulation of gene expression by photocrosslinking
oligonucleotide containing of carbazole
Atsuo Shigeno1, [email protected], Sakamoto Takashi1, kenzo
Fujimoto1,2. (1) Department of Materials Science, Japan Advanced
Insutitute of Science and Technolgy, Nomi, Ishikawa 923-1292, Japan (2)
Research Center for Bio-Architecture, Japan Advanced Insutitute of Science
and Technolgy, Nomi, Ishikawa 923-1292, Japan
Antisense method based on specific binding between Antisense
oligonucleotide (AS-ODN) and complementary target RNA can
regulate not only gene expression by specific binding with mRNA
but also micro RNA functions by specific binding with RNAinduced silencing complex. Therefore, the antisense method had
been well studied as drugs for gene therapy and tools for study on
micro RNAs. Until now, some functional AS-ODNs, such as highly
stable, high affinity and stimuli responsible AS-ODNs have been
already reported. Especially, photo-responsible AS-ODN has a
great potential for organ specific photodynamic antisense therapy.
However, unexpected photodamages were also caused because its
low photo-responsibility of present photo-responsible AS-ODNs.
In this study, 3-cyanovinylcarbazole modified nucleoside that
can effectively photo-crosslink to specific pyrimidine base in the
complementary RNA strand was adopted as photo-responsible
nucleotide in AS-ODN. Potential of the novel photo-responsible
AS-ODN was investigated from its sequence selectivity, target
generality and binding ability for target mRNAs.
100
Functional expression and characterization of a novel xylose
isomerase in Saccharomyces cerevisiae
Nobuhiko Muramoto1, Risa Nagura1, Moriya Ohkuma2, Shigeharu
MORIYA3. (1) Laboratory of Biotechnology, TOYOTA Central R&D
Labs., Inc., Nagakute, Aichi 480-1192, Japan (2) Bio Resource Center,
RIKEN Tsukuba Institute, Wako, Saitama 305-0074, Japan (3) Molecular
& Informative Life Science Unit, RIKEN Advanced Science Institute,
Yokohama, Kanagawa 230-0045, Japan
Saccharomyces cerevisiae, which is a promising candidate for
biomass ethanol production, is unable to metabolize xylose.
Although heterologous expression of xylose isomerase (XI) enables
to endow S. cerevisiae with the ability to utilize xylose, only a
few XI genes have been functionally expressed in S. cerevisiae.
We successfully cloned a novel class of XI genes from protists
resident in termite hindgut. Amino acid sequences encoded by
these genes have low sequence similarities to already known XIs.
One of these genes was functional in S. cerevisiae with high XI
activity. Remarkably, the Km value of the novel XI is much lower
than that of the XI from Piromyces sp. strain E2. The recombinant
yeast strain which introduced the novel XI gene could grow on
xylose faster than the strain harboring XI gene from Piromyces.
Furthermore, the recombinant strain produced ethanol from a
mixture of glucose and xylose with high yield.
BIOT 245
Decisional tool in supporting cost-effective bioprocess
development
Ying Gao1, [email protected], Sofia Simaria2, Suzy Farid2,
Turner Richard1. (1) Bioprocess Development, MedImmune Ltd.
Cambridge, United Kingdom (2) Department of Biochemical Engineering,
University College London, United Kingdom
With increasing pressures to meet the tight financial constraints
and to win the competition in the biopharmaceutical market,
companies are facing the challenges for the fast realisation of cost
effective and high yielding processes. This presentation describes
a decisional tool that integrates a process economics model with
uncertainty analysis and optimisation algorithms for the evaluation
of alternative downstream processing strategies, considering the
trade-offs between process operation and cost of goods (COGs).
Case studies are used to illustrate the application of the decisional
tool in evaluating alternative downstream process flowsheet
utilising newer chromatography resins and novel separation
techniques such as mixed-mode chromatography, precipitation,
etc, to assist decision-making during process development in
selecting the most efficient and cost-effective processes. Alternative
manufacturing strategies for both single and multi-product
manufacturing scenarios have also been investigated considering a
range of products with various titre and product demands.
Key words: Decisional tool, process economic analysis, alternative
bioseparation techniques, multi-product facility, capacity
utilisation.
BIOT 246
Design of modified hydrolytic enzymes for sugar production
from switch grass
Debra A. Clare, [email protected], Ziyu Wang, Jay J. Chen.NC
State University, Raleigh, NC 27695, United States
Hydrolytic enzymes, such as cellulase/β-glucosidase are commonly
used in the biofuels industry. This work addresses the possibility
for creating modified cellulase (mcellulase) and β-glucosidase
(mβGSD) prototypes, using three different immobilized proteases.
The experimental goal was focused on generating multiple,
smaller sized mcellulase/mβGSD molecular species, such that
high catalytic capabilities were retained. After cleavage, (i) the
degree of hydrolysis was determined (OPA assay), (ii) SDSPAGE protein banding patterns were visualized, and (iii) residual
cellulase and βGSD activities were measured using filter paper
and cellobiose substrates, respectively. The method for generating
functional mcellulase/mβGSD variants was optimized. This study
describes a simple model system for creating mcellulase/mβGSD
hydrolases which may exhibit improved catalytic efficiencies due to
increased accessibility to recalcitrant biomass substrates (cellulose/
hemicellulose). Ultimately, the goal of this research endeavor
is aimed at testing these methods/approaches in practical “real
world” applications using switch grass and/or woody biomaterials.
BIOT 247
Evaluation of alternative instruments for cell quantification of
single cell culture, clumpy culture, and microcarrier culture
Divya Harjani, [email protected], Ravinder Bhatia.API - Large
Molecule, Johnson & Johnson Pharmaceutical Research & Development,
Spring House, PA 19477, United States
Viable cell density and culture viability are critical process parameters
in cell culture. Accurate and reproducible quantification of the cells
in your process is critical to batch success. With the evolution of
technology, several instruments have emerged on the market that
utilize various techniques for cell quantification. In this study, three
instruments were evaluated to quantify cells that presented varying
morphologies. A CHO cell line was used as the standard singlecell suspension culture. It was compared to a non-uniform, clumpy
culture. Finally, cells grown on microcarriers were also evaluated.
The CEDEX currently stands as the gold standard for quantification
of single cell suspension culture; however, instrument design and
software setup present challenges in the quantification of clumpy
cell lines and microcarrier culture. Using the popular Trypan Blue
Exclusion method, the CEDEX captures microscopic images of the
cell sample and a pre-designed algorithm accepts items of a certain
size as cells and items above and below that size as non-cellular,
excluding those in the cell count. As a result, Clumpy cells are often
disregarded by the software as non-cells, and are not quantified
accurately. Microcarriers, on the other hand, can clog the intricate
tubing system of the instrument unless they are clarified from the
sample first.
To address these challenges, the NucleoCounter – 100 and
NucleoCounter – 3000 were tested. The mechanisms of action of
these instruments provide a biological advantage that is favorable
for the quantification of clumpy and microcarrier cell culture. The
NucleoCounter NC-100 has an integrated fluorescent microscope
that detects the dye Propidium Iodide (PI), which binds to
DNA. The details of these instruments, as well as advantages
and disadvantages for each cell line are discussed. Using the data
generated from this study, we can make a recommendation for cell
quantification for each of these cell types.
BIOT 248
Recombinant silk materials from sea anemone
Yun Jung Yang1, Yoo Seong Choi2, Dooyup Jung1, Hyung Joon Cha1,
[email protected]. (1) Department of Chemical Engineering, Pohang
University of Science and Technology, Pohang, Republic of Korea (2)
Department of Chemical Engineering, Chungnam National University,
Daejon, Republic of Korea
Under certain stimulus, sea anemone stretches and shrinks its
body rapidly. We assumed that silk or elastin-like protein involves
this reaction and found that there are unique repeats of decamer
mainly consists of glycine and proline more than 50% of its whole
amino acids. Through immunohistochemistry, we proved that the
unique repeat sequence is distributed along with skin, especially
at their tentacles. This discovered silk-like protein (30 kDa) was
successfully expressed in Escherichia coli, spun by wet spinning
method, and performed tensile test to determine mechanical
properties. Its properties, in terms of strength and extensibility,
exceeded to elastin or resilin. To improve the properties (strength,
extensibility, stiffness and toughness), we constructed repeated
60 KDa of silk-like protein. Through this approach, strength of
protein was able to compete against synthetic rubber. This 60 kDa
silk-like protein also showed outperformed stiffness compared to
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to NADH. Given that NADH is a preferred redox cofactor, this
switch in coenzyme specificity is valuable in engineering microorganism(s) to produce 3-HP.
BIOT 249
Evaluation of ethanol production from renewable cellulosic
resources using process simulation tools
Demetri Petrides, [email protected], Charles Siletti.Intelligen,
Inc., United States
BIOT 251
Examining the transcriptional response of overexpression
anthranilate synthase（ASαβ) in Catharanthus roseus hairy
roots
Jiayi Sun, [email protected], Christie A.M. Peebles.
Department of Chemical and Biological Engineering, Colorado State
University, Fort Collins, CO. 80526, United States
Over the past three decades there has been intense investigation on
the development of fuel producing processes that are based on the
use of renewable agricultural materials as feedstock. This activity
is driven primarily by the quest for fuel self-reliance and carbon
oxides emission reductions. The main effort has been concentrated
on bio-ethanol and bio-diesel which have been shown to give motor
engine performance similar to that of conventional petroleum
based fuels. In addition to product characteristics, however, process
economics play an equally important role in any successful product
commercialization. In this work, realistic process simulation
models have been developed in order to analyze the economics of
corn-stover to ethanol conversion. This presentation will illustrate
how such models can guide R&D work and facilitate process
optimization.
Catharanthus roseus produces a variety of terpenoid indole
alkaloids (TIAs) which include the commercially valuable anticancer drugs vinblastine and vincristine. We are interested in the
metabolic engineering of the TIA pathways and the effects this has
in C. roseus hairy roots.
The biosynthetic pathway leading to the TIAs involves the coupling
of secologanin from the terpenoid pathway with tryptamine from
the indole pathway by strictosidine synthase. Previous work on this
system has shown that over-expression of a feedback insensitive
anthranilate synthase (ASαβ) under the control of an inducible
promoter increased the levels of tryptophan, tryptamine and
ajmalicine, but decrease the levels of lochnericine, hörhammericine
and tabersonine. We are interested in the effect of overexpressing
one gene on the other genes in TIA pathway. We will discuss the
transcriptional response of all known TIA genes and regulators
through RT-qPCR at various time points after induction of ASαβ.
BIOT 250
BIOT 252
Investigation of complex coacervation using recombinant
mussel adhesive proteins
Overexpression and characterization of putative undecaprenyl
phosphate galactose-1-phosphate transferase from a pathogen
Vibrio cholerae
Seonghye Lim, [email protected], Hyung Joon Cha.Department
of Chemical Engineering, Pohang University of Science and Technology,
Pohang, Kyoungbuk 790-784, Republic of Korea
Mussels inhabit seashore by attachment themselves using their
adhesive proteins. Mussel adhesive proteins (MAPs) have various
forms of foot proteins which are stockpiled in vacuole with highly
condensed liquid phase. Coacervation process has been suggested
to explain the condensation of protein solution. Complex
coacervation is liquid/liquid phase separation where the oppositely
charged polyelectrolytes meet, followed by separation of condensed
and diluted phase. Moreover, condensed coacervates phase has
benefit for underwater adhesion in that is non-dissolved in watery
phase and has low interfacial tension. However, coacervation
process has been regarded as impossible phenomenon in mussel
adhesion system because acidic partner was not found for basic
MAPs. In this study, we present newly found acidic partner and
confirmation complex coacervation in mussel adhesion using
several recombinant MAPs.
102
Chang Sup Kim1, [email protected], Jae Kyung Sohng2, Hyung
Joon Cha1. (1) Department of Chemical Engineering, Pohang University
of Science and Technology, Pohang, Kyungbuk 790-784, Republic of Korea
(2) Department of Pharmaceutical Engineering, Sun Moon University,
Chungnam 330-150, Republic of Korea
Vibrio cholerae is a gram-negative bacterium with a polar flagellum
that causes cholera in humans. They produce a various types of
lipopolysaccharide (LPS) on outer membrane. While the lipid A and
core OS of different V. cholerae serogroups show similar structures,
O-antigen present a distinct structure. Of V. cholerae serogroups,
O-antigen of V. cholerae O1 is controlled by ABC transporterdependent pathway. O-antigens formed by ABC transporterdependent pathway shows linear structures and comprise three
parts: primer, adaptor, and repeat unit domain. To date, O antigen
synthesis controlled by this pathway is initiated by a homolog of
WecA, the enzyme which catalyze the transfer of GlcNAc-1-P
from UDP-GlcNAc (donor) to undecaprenyl phosphate (acceptor).
However, it speculates that an initial O-antigen synthesis of V.
cholerae O1 is regulated by a homolog of WbaP (undecaprenyl
phosphate galactose-1-phosphate transferase). Here, we report
purification and characterization of putative undecaprenyl
phosphate galactose-1-phosphate transferase from a pathogen
Vibrio cholerae
BIOT 253
Chaperone co-expression increased periplasmic expression of
organophosporus hydrolase in Escherichia coli
Dong Gyun Kang1, Im Gyu Kim1, Chang Sup Kim1, redcskim@
postech.ac.kr, Suk Soon Choi2, Geunbae Lim3, Hyung Joon Cha1. (1)
Department of Chemical Engineering, Pohang University of Science and
Technology, Pohang, Kyungbuk 790-784, Republic of Korea (2) Department
of Biological and Environmental Engineering, Semyung University, Jecheon,
Republic of Korea (3) Department of Mechanical Engineering, Pohang
University of Science and Technology, Pohang, Kyungbuk 790-784, Republic
of Korea
Twin arginine translocation (Tat) pathway has advantage of
secreting protein in periplasm after it is folded in cytoplasm. When
organophosphorus hydrolase (OPH) is expressed with Tat signal
sequence in Escherichia coli, inclusion body in cytoplasm is a
dominant form. Therefore, whole cell activity is relatively low. In
the present work, we investigated a strategy for overcoming this
problem in a whole cell system by enforcing periplasmic secretion
of OPH through chaperone co-expression. We co-expressed
molecular chaperone including GroEL/ES with OPH. We found
significant increase of OPH in a soluble form compared to that
without chaperone and this might be due to increased protein
folding. Furthermore, whole cell OPH activity of chaperone
co-expressing cells was about 20 times greater than that of nonexpressing cells. Chaperone may successfully assist in enhancement
of whole cell OPH activity
BIOT 254
Biosynthesis of morphinan and berberine benzylisoquinoline
alkaloids in Saccharomyces cerevisiae
Stephanie Galanie1, [email protected], Kate Thodey2, Christina
D Smolke2. (1) Department of Chemistry, Stanford University, Stanford,
CA 94305, United States (2) Department of Bioengineering, Stanford
University, Stanford, CA 94305, United States
Benzylisoquinoline alkaloids (BIAs) are medically useful natural
products. For many BIAs, including morphine, no commercially
viable syntheses exist and supply is limited to extraction from
plants, particularly opium poppy. A BIA production platform that
is less resource intensive and less affected by environmental and
political climates is desirable.
Previously, researchers engineered Saccharomyces cerevisiae
and Escherichia coli to convert dopamine or norlaudanosoline
to reticuline, an important branch point intermediate in BIA
biosynthesis. A major challenge in heterologous BIA biosynthesis
is obtaining sufficient activity in the microbial host from the
numerous plant cytochrome P450s. For example, salutaridine
synthase is a morphinan P450 enzyme that catalyzes C-C phenol
coupling to convert reticuline to salutaridine. The human
P450 CYP2D6 can perform this reaction non-regiospecifically.
However, using the human enzyme in the reconstructed pathway
limits flux through the morphinan branch, likely because CYP2D6
does not optimally interact with plant-derived enzymes, limiting
productivity.
We will present ongoing efforts to advance a microbial platform to
synthesize high-value morphinan and berberine BIAs. Specifically,
efforts towards (1) optimizing enzyme expression levels, (2)
spatially engineering pathway enzymes, and (3) developing
generalizable strategies for functional expression of plant P450s in
a yeast chassis will be described.
BIOT 255
Mussel adhesive protein-based multicomponent artificial
extracellular matrix mimics for bone tissue engineering
Bong-Hyuk Choi, [email protected], Hyung Joon Cha.
Department of Chemical Engineering, Pohang University of Science and
Technology, Pohng, kyungbuk 790-784, Republic of Korea
The concept of artificial extracellular matrix (ECM) is very
important approach in tissue engineering fields due to its significant
biological activities. However, it is required that development
of facile and efficient technique for multicomponent coating
on artificial ECM surface. Recently, we reported artificial ECM
mimics based on fusion of mussel adhesive protein (MAP) with the
biofunctional ECM peptides. Adhesive properties of MAP enabled
efficient immobilization of ECM peptides without any protein and/
or surface modifications, which significantly enhanced cellular
behaviors on each ECM mimics. Here, we easily constructed
multicomponent artificial ECM environment through simple
combination of several artificial ECM mimics. Diverse biological
activities such as adhesion, proliferation, and differentiation on
artificial ECM mimic mixture-coated surfaces were investigated
for several bone cell lines. We found that multicomponent artificial
ECM mimics showed superior abilities on cells to single component
ECM mimics. Thus, multicomponent artificial ECM based on
biofunctional peptide-conjugated MAP can be successfully applied
in tissue engineering.
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previous 30 kDa protein and tendon collagen. As for extensibility,
it was roughly analogous to Kevlar, a synthetic fiber.
Template directed reversible photochemical ligation for DNA
nanotechnology
Shigetaka Nakamura1, [email protected], Kenzo
Fujimoto1,2. (1) Department of Materials Science, Japan Advanced
Institute of Science and Technology, Nomi, Ishikawa 923-1292, Japan (2)
Department of Research Center for Bio-Architecture, Japan Advanced
Institute of Science and Technology, Nomi, Ishikawa 923-1292, Japan
Gene manipulation rely on enzymes, such as ligase, DNA/RNA
polymerase, and restriction enzymes, the conditions for gene
manipulation are largely restricted by the pH, temperature, and salt
concentration of the media. Various efforts have been made to overcome
the restrictions of gene manipulation technologies. We developed
novel nucleobases analogues having photoreactivity for dimerization
with adjacent nucleobases by longer wavelength irradiation. This
enable specific photodimerization of the photoreactive nucleobase
and adjacent nucleobases without undesired dimerization of other
natural nucleobases, and also enables phototriggered ligation between
DNA strands via photo-dimerization of the photoreactive nucleobase
analogues for the photoligation of nucleic acids and its applications for
DNA nanotechnology.
BIOT 257
Development about photochemical DNA and RNA crosslinking
via 3-cyanovinylcarbazole
Satomi Kishi, [email protected], Daiki Futamura, Kaoru Hiratsuka,
Takashi Sakamoto, Kenzo Fujimoto.Department of Materials Science,
Japan Advanced Institute of Science and Technology, Nomi, Ishikawa 9231292, Japan
We have developed that a modified oligodeoxynucleotide (ODN)
containing 3-cyanovinylcarbazole nucleoside (CNVK) can be
photoreversibly crosslinked with cytosine or thymine via UV
irradiation at two different wavelengths, and perform the reversible
DNA photocrosslinking via [2+2] cycloaddition between CNVK
and cytosine or thymine on the irradiation condition of seconds.
We have also developed photo-induced artificial DNA and
RNA editing based on our ultrafast reversible DNA and RNA
photocrosslinking.
BIOT 258
Biological fixation of carbon dioxide and enhanced production
of organic compounds by inducible overexpression of the
carbonic anhydrase in Rhodobacter sphaeroides
104
Ju-Yong Park1, [email protected], Yang-Hoon Kim2, Jiho Min1. (1)
Graduate school of semiconductor and chemical engineering, Chonbuk
National University, Jeonju, Jeonbuk 561-756, Republic of Korea (2)
Department of Microbiology, Chungbuk National University, Cheongju,
Chungbuk 361-763, Republic of Korea
Carbon dioxide is a major cause for the global warming, thus
the reduction of the CO2 is needed. Carbonic anhydarse (CA
; EC 4.2.1.1) is as zinc-containing metalloenzyme catalyzing
the reversible hydration of CO2. In Purple non-sulfur bacteria,
intracellular CA enhances the rate of CO2 to HCO3- conversion,
for fixation by phosphoenolpyruvate carboxylase. In this study,
to induce over-expression of CA, Rhodobacter sphaeroides was
used as a recipient strain for transformation with the plasmid
pBBR1mcse-2. This result showed that cells used more CO2 and
enhanced the production of organic compounds. The function
of CA in R. sphaeroides would accelerate the biocarbonate-CO2
conversion in the intracellular. Therefore, this study will prove
useful in efforts to improve CO2 fixation and photosynthetic
ability in this species for a variety of biotechnological applications.
BIOT 259
Preparation and characterization of TiO2 nanoparticles and
their utilization for the degradation of caramel pigment in
sauce wastewater
Li Xiaolong1,2, Zhang Fengqin2, Ma Chao1, He Nongyue1,
[email protected]. (1) State Key Laboratory of Bioelectronics, Southeast
University, Nanjing, Jiangsu 210096, China (2) Hunan key Laboratory of
Green Packaging and Application of Biological Nanotechnology, Hunan
University of Technology, Zhuzhou, Hunan 412008, China
TiO2 photocatalysis is a well- known technology for removing
organic and inorganic pollutants from wastewater. In this study, N,
C, and Cu doped TiO2 nanoparticles (NPs) were prepared by an
ultrasonic-assisted sol-gel method. TiO2 NPs were characterized
by X-ray diffraction (XRD), scanning electron microscopy (SEM)
， energy dispersive spectroscopy (EDS) measurements and UVvis absorption spectroscopy. Photocatalytic removal of caramel
pigment from sauce wastewaster using different photocatalysts
were conducted. Compared to un-doped TiO2, the doped TiO2
have the improved absorbance from 260 nm to 400 nm. All catalysts
showed the ability to catalyze the photodegradation of caramel
pigment from an aqueous solution. In terms of catalytic activity the
synthesized TiO2 NPs were almost comparable to the commercial
TiO2 photocatalyst. In addition, the doped TiO2 showed higher
catalytic activity, especially in the case of Cu doped TiO2 NPs.
BIOT 260
Cellular mechanisms underlying toxicity and transport of
hydrocarbons in Saccharomyces cerevisiae
Hua Ling, Binbin Chen, Wei Suong Teo, Hongxin Zhao, Pei Yu Lim,
Hazarki Yaohari, Chi Bun Ching, Susanna Leong, Matthew Wook
Chang, [email protected] of Chemical and Biomedical
Engineering, Nanyang Technological University, Singapore, Singapore
637459, Singapore
Despite the widespread presence of hydrocarbons, including
alkanes, in the environment, there is a lack of understanding of
interactions between hydrocarbons and biological systems. In this
study, we aimed to elucidate molecular mechanisms underlying
toxicity, transport, and accumulation of alkanes in Saccharomyces
cerevisiae. Our results show that medium chain alkanes significantly
decreased the viability of S. cerevisiae without causing apparent
damage to the cell surface. Interestingly, upon exposure to alkanes,
intracellular medium chain alkanes were detected in S. cerevisiae,
implying that some alkanes may be diffused and/or transported
into S. cerevisiae. From microarray-based transcriptome analysis,
we identified cellular mechanisms potentially associated with
alkane toxicity and transport in S. cerevisiae. Further, we show
that the expression of exogenous efflux transporters decreased the
toxicity of hydrocarbons over 5-fold. Overall, this study provides
new insights into molecular and cellular mechanisms underlying
the toxicity, transport, and accumulation of alkanes in S. cerevisiae.
BIOT 261- Withdrawn
BIOT 262
Probing product inhibition of processive and nonprocessive
cellulases
Lintao Bu1, [email protected], Mark R. Nimlos1, Michael
R. Shirts3, Michael E. Himmel2, Michael F. Crowley2, Gregg T.
Beckham1. (1) National Bioenergy Center, National Renewable Energy
Laboratory, Golden, CO 80401, United States (2) Biosciences Center,
National Renewable Energy Laboratory, Golden, CO 80401, United
States (3) Department of Chemical Engineering, University of Virginia,
Charlottesville, VA 22904, United States
Product inhibition significantly impacts the efficiency of
cellulose deconstruction by cellulase enzymes, but experimental
measurements of product inhibition constants vary by orders of
magnitude, and there is little consensus on the importance of this
phenomenon. Deeper understanding of product inhibition and
strategies to mitigate this issue will enable more efficient utilization
of plant biomass as a sustainable energy resource. Here we examine
the impact of product binding on both processive and nonprocessive
cellulases by calculating the binding free energy of cellobiose to the
catalytic domain of representative processive and nonprocessive
enzymes from glycoside hydrolase (GH) families 6 and 7 using
steered molecular dynamics (SMD). Several point mutations on
the key binding residues of the enzymes identified from the SMD
trajectories were also made computationally to study the binding
free energy changes during the product expulsion process. Our
goal is to engineer the enzymes to accelerate the product expulsion
process and improve the efficiency of biomass conversion.
BIOT 263
Bioconversion of glycerol surplus into L-tryptophan using
tryptophan-indole lyase
Quang T Do1, [email protected], Robert S Phillips1,2. (1)
Department of Chemistry, University of Georgia, Athens, Georgia 30602,
United States (2) Department of Biochemistry and Molecular Biology,
University of Georgia, Athens, Georgia 30602, United States
Resulted from the abundance of glycerol surplus from biodiesel
processing, our interest is to utilize the glycerol metabolism, which
intercepts glycolysis in the generation of pyruvate (Scheme 1),
in an expression system for tryptophan-indole lyase to develop
a convenient one-pot biosynthesis for L-tryptophan (Scheme 2).
Using Escherichia coli BL21 (DE3), with plasmid pET15b:tnaA, we
were able to demonstrate, as a proof-of-concept, that glycerol was
successfully and conveniently converted into L-tryptophan.
BIOT 264
Robust comparison of data from high-density mammalian cell
perfusion cultures
Meile Liu, [email protected], Chetan T Goudar.Cell Culture
Development, Global Biological Development, Bayer HealthCare, Berkeley,
CA 94710, United States
Mammalian cell perfusion bioreactors are characterized by long
cultivation times, often in excess of 100 days. Since perfusion
bioreactors operate at steady-state, the long cultivation duration
results in a rich data set of prime variables and their associated
cell specific rates for characterizing cell growth, metabolism and
protein production. Comparison of cell performance between
bioreactor runs is routinely performed to evaluate cell bank
and bioreactor operational consistency, and to compare the
performance of laboratory and manufacturing-scale bioreactors.
In addition, process improvements related to medium composition
and bioprocess variables are typically evaluated over the course of
a perfusion experiment which necessitates comparison between
control and test conditions. There is thus a need for robust analysis
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BIOT 256
BIOT 265
Elucidating transcriptional regulation of terpene production
in S. cerevisiae
Sarah Rodriguez1, [email protected], Jay Keasling2.
(1) Molecular and Cellular Biology, UC Berkeley, Berkeley, CA 94720,
United States (2) Joint Bioenergy Institute (JBEI), Emeryville, CA 94608,
United States
Recently sesquiterpenes, terpenes generated from the precursor
farnesyl-pyrophosphate, have revealed themselves as potential
biofuel candidates. In the widely utilized industrial fermentation
host, S. cerevisiae, the pathway that generates terpenes can be utilized
for the production of sesquiterpene metabolites with the addition
of heterologous terpene synthases. Having exhausted obvious
targets to increase sesquterpene production we hypothesized that
over-expression the heterologous enzymes which convert acetylcoA to mevalonate, would divert more flux through the mevalonate
pathway. Over-expression of these enzymes demonstrated ~500
% increase in mevalonate but a ~30% decrease in sesquiterpene.
employed the global profiling tools of transcriptional microarrays
and metabolomics to determine host responses occurring due
to accumulation of mevalonate. Obtaining an understanding
of mevalonate pathway regulation in S. cerevisiae is key for the
development tools to work with or overcome host regulation.
BIOT 266
Application of flux balance analysis (FBA) to mammalian cell
perfusion cultures
Richard Biener1, Karthik P Jayapal2, [email protected],
Chetan T Goudar2. (1) Department of Natural Sciences, University
of Applied Sciences Esslingen, Esslingen, Germany (2) Cell Culture
Development, Bayer HealthCare, Berkeley, CA 94710, United States
Metabolic flux analysis is being increasingly used to characterize
mammalian cell physiology and metabolism. Experimental
approaches have included both metabolite balancing and isotope
labeling approaches with glucose and glutamine as the primary
carbon sources. These studies have typically involved overdetermined systems where excess experimental data have been
collected and used to characterize measurement error and to
evaluate the appropriateness of the chosen biochemical network.
Alternatively, FBA has been typically used to characterize large
under-determined networks and several hypothesis around cell
behavior have been used to arrive at the final flux distribution in
the metabolic network. In the present study, we compare results
from flux analysis in an over-determined system with those
obtained from FBA using different assumptions regarding cell
physiology and metabolism. Accuracy of the experimental data
and associated specific rates was verified using the consistency
index and a comparison with the FBA flux data helped validate
which of the hypothesis were consistent with the experimental
findings. Results from this comparison should help future FBA
studies on large bioreaction networks where limited experimental
data are available.
BIOT 267
Gene expression profiling during fed-batch cultivation of
mammalian cells
Meile Liu, [email protected], Chetan Goudar.Cell Culture
Development, Global Biological Development, Bayer HealthCare, Berkeley,
CA 94710, United States
These results hint at the possibility of transcriptional regulation
or allosteric regulation of the mevalonate pathway. We have
106
Recent advances in cell line and cell culture process development
have resulted in high monoclonal antibody yielding fed-batch
processes both for early clinical material production and subsequent
commercial manufacturing. These higher productivities are often
associated with reduced development time which further highlights
the importance of recent progress in this area. However, process
robustness and product quality concerns still persist which in the
extreme case can result in lot rejections thereby compromising
product supply to patients. While traditional macroscopic and
empirical approaches to process understanding are critical,
approaches based on first principles have the potential to alter
our understanding of cellular physiology and metabolism in a
fundamental way and can contribute towards the development of
more robust bioprocess with minimal product quality deviations.
In this context, we have characterized gene expression profiles
from multiple mammalian cell fed-batch experiments to obtain
information related to central carbon metabolism, amino acid
and vitamin metabolism, and the glycosylation pathways. Time
courses of gene expression data helped understand the dynamics
over the course of an experiment while comparison across multiple
bioreactor runs helped quantify the impact of process changes on
gene expression profiles. Information from these studies augments
the routine data set of cell culture variables and greatly enhances
our understanding of cellular response to bioprocess changes at the
genetic level.
BIOT 268
Synthesis of bioactive 3D porous silica nanofiber scaffolds by
electrospinning method
Yui Whei Chen-Yang1,2, [email protected], Wei-Ning Yang4,
Pei Hsin Hsieh2, Yen-Kuang Li1, Ting-Yu Chin2,3, Po-Zen Fan Jen3. (1)
Department of Chemistry, Chung Yuan Christian University, Chung-Li,
Taiwan Republic of China (2) Center for Nanotechnology, Chung Yuan
Christian University, Chung-Li, Taiwan Republic of China (3) Department
of Bioscience Technology, Chung Yuan Christian University, Chung-Li,
Taiwan Republic of China (4) Master Program in Nanotechnology, Chung
Yuan Christian University, Chung-Li, Taiwan Republic of China
In this study, the porous silica nanofibers were prepared by the
electrospinning process using polyvinyl pyrrolidone (PVP) as
a thickener. After heat treatment to remove the PVP, a threedimensional porous silica nanofibers (SNF) were obtained. The
silica nanofibers were further modified by the biologically active
functional groups to form the biological scaffolds. The as-prepared
three-dimensional SNF were characterized by FT-IR, TGA, BET
and solid-state 29Si-NMR measurements, and the morphology of
nanofibers was studied by SEM, AFM and XRD measurements. In
addition, the functionalized porous silica nanofibers were used as
three-dimensional scaffolds for a neural stem cells to investigate
the effect of the scaffold. The results showed that the modified
porous silica nanofibers were not only non toxic to the stem cell
but also good to the cell proliferation. This study indicates that the
functionalized porous silica nanofibers are promising biological
scaffold materials for stem cell system.
Keyword: Electrospinning, Nanofiber, Biological scaffolds, SiO2
Acknowledgement. The authors gratefully acknowledge National
Science Council, Taiwan (ROC) (99-2632-M-033-001-MY3) and
Chung Yuan Christian University, Taiwan, ROC (CYCU-98-CRCH) for supporting the research work.
BIOT 269
Development and implementation of a lab-scale data
acquisition and monitoring system
Terrance W Carone1, [email protected], Greorgy M
Hartnett2, Susan F Abu-Absi1. (1) Manufacturing Science and Technology:
Upstream, Bristol-Myers Squibb Company, East Syracuse, NY 13057,
United States (2) Department of Biomedical Engineering, University of
Rochester, Rochester, NY 14627, United States
Laboratory-scale bioreactor studies are widely used in the
biopharmaceutical industry to generate process robustness data.
Small-scale studies that employ a design of experiment (DOE)
approach can require many sets of experiments to achieve high
resolution data for multiple parameters, which can be time
consuming and difficult to execute. Therefore, laboratory space
and equipment should be configured to reduce human error and
enable an efficient work flow. One approach is to implement a
system capable of automatically controlling bioreactor inputs,
outputs, data collection, data display and setpoint adjustments.
The objective of this project was to develop a communication
system that would allow several analytical devices to exchange data
to simplify the lab-scale bioreactor sampling process and improve
monitoring and control. Open Process Control technology and
Visual Basic for Applications were used to create a data acquisition
and controller application in Microsoft Excel allowing users to
quickly acquire and electronically transfer data from multiple
bioreactors into a spreadsheet. Real-time data is acquired and can
then be used to directly control bioreactor parameters through a
MFCS/win client or to monitor the process day-to-day through
graphical data plots and charts. The data acquisition system
provides a user with the ability to make automatic process changes
such as temperature or agitation shifts throughout the experimental
cycle as determined by batch recipes.
Development and implementation of this MFCS/win data
communication system resulted in a more efficient laboratory
process. Use of VBA, MS Excel and OPC software to control and
monitor bioreactors in real-time enabled data to be collected and
bioreactor controls adjusted throughout the experiment with little
human interaction. Use of data acquisition systems, feedback
control loops and PAT concepts to control lab-scale bioreactors
is part of the QbD principles that are being implemented in
biopharmaceutical manufacturing facilities to design, analyze and
control large scale processes.
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of steady-state data from perfusion bioreactors. In this study, we
present results from the application of a multitude of statistical
methods for comparing data sets from perfusion bioreactors.
Specifically, the limitations of conventional comparison approaches
and the importance of accounting for measurement and
computational errors are highlighted. The results from this study
have implications both for process develompent, manufacturing
support, and commercial biotherapeutic production in mammalian
cell perfusion bioreactors.
Engineering Escherichia coli to produce dihomomethionine
Aram Kang, Wendy Chen, Chi Bun Ching, Susanna Leong, Matthew
Wook Chang, [email protected] of Chemical and
Biomedical Engineering, Nanyang Technological University, Singapore,
Singapore 637459, Singapore
This study aimed to metabolically engineer Escherichia coli
to produce dihomomethionine (DHM). Towards this aim,
genes involved in DHM biosynthesis pathways in plant cells
were introduced in E. coli and their expression was improved
through codon optimization and signal peptide truncation.
To increase DHM production level, the constructed metabolic
pathway was optimized as follows. First, optimal strength of
promoters was determined by steady-state modeling, and various
combinations of promoters were experimentally tested. Second,
methionine-metabolizing pathways were sequentially disrupted
by gene-knockout mutagenesis. Third, metabolic fluxes in the
knockout mutants were adjusted by in silico flux balance analysis.
Further, culture conditions including temperature and medium
compositions were optimized. In particular, effects of methionine
supplementation on the production yield were determined. This
study represents the first report of DHM production in microbes
and provides new insights into the feasibility of DHM production
in microbial hosts.
throughput enzyme assay were considered “positive hits” as
determined by ANOVA and t-test analyses. The responses to these
agents were confirmed by repeat experimentation in 24 well plates.
Based on the magnitude of response and economic feasibility,
sodium butyrate, sodium propionate, and dimethyl sulfoxide
(DMSO) were chosen for verification in a four vessel 1.2 L parallel
bioreactor system run under identical process parameters. While
the viable cell density (VCD) for the supplemented reactors was
3-4 fold lower than the control, GUS output in the supplemented
reactors nearly doubled. These results demonstrate that a two level
static batch approach to screening in multi-well plates can be a
viable strategy to improve protein output in benchtop bioreactor
systems.
*Acknowledgements: This research was supported by Critical Path
funding from the U.S. Food and Drug Administration (Proposal
#1499) to G.R.J.
Disclaimer: The findings and conclusions in this article have not
been formally disseminated by the Food and Drug Administration
and should not be construed to represent any Agency determination
or policy.
Screening of supplements to increase output of a model
therapeutic enzyme protein and qualification in a parallel
bioreactor system
Chikkathur Madhavarao1, Cyrus Agarabi2, [email protected].
gov, Maliha Khan1, Claudia Chen1, Howard Anderson1, Gibbes Johnson1.
(1) Office of Biotechnology Products- Division of Therapeutic Proteins, US
Food and Drug Administration, Bethesda, Maryland 20892, United States
(2) Office of Testing and Research- Division of Product Quality Research,
US Food and Drug Administration, Silver Spring, MD 20993, United States
Maximizing the yield of therapeutic enzymes produced in
bioreactors is critical to product development, meeting market
demand and preventing drug shortages. To improve protein
output, vast arrays of compounds were screened by supplementing
a commercially available chemically defined medium for Chinese
Hamster Ovary (CHO) cells that secrete the glycosylated
recombinant human enzyme beta-glucuronidase (GUS). More
than 400 supplements which included carbon and energy sources,
dipeptides, hormones, metabolic effectors and trace metals were
screened in 96-well phenotypic microarray (PM) plates. Increased
GUS activity levels determined via a robust and sensitive high
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BIOT 275
Exploring the stress-response and resistance mechanisms to
fuel candidates in Saccharomyces cerevisiae
Development of a robust S. cerevisiae strain for the high-level
synthesis of pyrones
Zain Y Dossani1,2, [email protected], Jay D Keasling1,2, Aindrila
Mukhopadhyay1,2. (1) Fuels Synthesis, Joint BioEnergy Institute,
Emeryville, California 94608, United States (2) Physical Biosciences
Division, Lawrence Berkeley National Laboratory, Berkeley, California
94720, United States
Javier Cardenas, [email protected], Nancy A. Da Silva.Department
BIOT 272
Many target compounds under consideration as biofuel candidates
are toxic to microorganisms, creating a problematic tradeoff
between yield and survival. Here, we aim to shed light on the
stress response induced in the yeast Saccharomyces cerevisiae, a
commonly engineered host for fuel production, in the presence
of terpene fuel candidates. Transcriptomic measurements in the
presence of toxic and non-toxic fuel compounds reveal both general
and specific stress responses. Additionally, a functional genomics
approach utilizing the S. cerevisiae knockout collection uncovers
hypersensitive and resistant strains. The combination of these two
approaches furthers our understanding of terpene-induced stress
responses and provides a framework for engineering more tolerant
strains that can achieve greater fuel yields.
Expression and recovery of immunotoxins from
Chlamydomonas reinhardtii chloroplast
BIOT 274
Lisa R. Wilken1, [email protected], Miller Tran2, Stephen Mayfield2,
BIOT 271
BIOT 273
Zivko L Nikolov1, [email protected]. (1) Biological & Agricultural
Engineering, Texas A&M University, College Station, TX 77843, United
States (2) Biological Sciences, University of California, San Diego, La Jolla,
CA 92093, United States
Chloroplast expression systems are promising platforms for
recombinant protein production. Chloroplasts provide an excellent
environment for proper folding of heterologous eukaryotic
proteins. Transgenic microalgae that grow non-photosynthetically
provide a protected intracellular space that is non-essential to cell
growth. We have recently demonstrated the ability of C. reinhardtii
chloroplasts to express a unique class of proteins, consisting of
antibody molecules genetically linked to Pseudomonas exotoxin A.
These proteins, termed “immunotoxins”, have the ability to target
cancerous cells and deliver the cell-killing toxin.
In this presentation, we report our efforts in expressing and
purifying two immunotoxins. Challenges related to downstream
processing and recovery of recombinant fusion proteins will
be presented. Specifically, we will discuss optimal conditions
for extraction and clarification of algal tissue and the required
pretreatment methods to address extract components that are
detrimental to immunotoxin activity and purification efficiency.
Engineering yeast for production of medium chain
hydrocarbons
Pei Yu Lim, Hazarki Yaohari, Hongxin Zhao, Matthew Wook Chang,
Susanna Su Jan Leong, [email protected] of Chemical and
Biomedical Engineering, Nanyang Technological University, Singapore,
Singapore 637459, Singapore
Biofuel production in microbial platforms using renewable sugar
feedstocks is a promising strategy that can potentially address
shortage of non-renewable petroleum fuels, which has become a
global problem today. The advancement of genetic and metabolic
engineering tools has opened the way for rational engineering of
microbes to produce a wide range of target products including
fuel molecules. In this study, we report the engineering of S.
cerevisiae for the production of saturated hydrocarbons, which
are essential components of petroleum fuels. We constructed a
pathway in S. cerevisiae that demonstrated efficient conversion of
medium chain fatty acids into alkanes via two steps (i.e. reduction
followed by decarbonylation) with uncompromised cell viability.
Protein engineering efforts are also underway to further improve
conversion yield to attain economic viable titers of the end product.
of Chemical Engineering & Materials Science, University of California Irvine, Irvine, CA 92697, United States
As petroleum-based feeds continue to be depleted, industries that
rely on them have an increased pressure to pursue biorenewable
means. As part of the Center for Biorenewable Chemicals
(CBiRC), we are investigating polyketide biosynthesis pathways
in Saccharomyces cerevisiae as a source of renewable carbon
precursors. By coupling enzyme engineering, enzyme expression
systems, and robust pathway engineering, our goal is to achieve
high-level synthesis of cyclic pyrones in S. cerevisiae. Initially, by
combining the most promising synthases with strains engineered
for improved expression of enzymes and precursors, we have
increased titer 15-fold and yield by 30-fold (g/g glucose). Continuing
to engineer S. cerevisiae’s pathways and enzyme expression systems
will lead to an attractive biocatalyst for the biorenewable chemicals
industry.
BIOT 276
Engineering Saccharomyces cerevisiae for the synthesis of
short-chain carboxylic acids using a type II FAS system
Ruben Fernandez-Moya, [email protected], Javier Cardenas,
Chris Leber, Nancy Da Silva.Department of Chemical and Biochemical
Engineering, University of California-Irvine, Irvine, CA 92697, United
States
The type I fatty acid synthase (FAS) is considered an efficient
biosynthetic enzyme for the production of fatty acids due to the
proximity of the active sites in a single large polypeptide. However,
this structure can also make manipulation of the enzyme difficult,
e.g. for the synthesis of shorter fatty acids. In contrast, the type II
FAS system is characterized by the use of discrete, monofunctional
enzymes and therefore is more flexible and easier to optimize
for the synthesis of fatty acids of defined length. In addition, the
intermediates of the FAS II system may be used in the synthesis
of other platform chemicals. We will present our studies on the
introduction of a bacterial FAS II pathway into Saccharomyces
cerevisiae to provide this flexibility and to increase the yield of
short chain carboxylic acids.
109
Tuesday Afternoon
BIOT 270
Characterizing and comparing secretome from breast cancer
and normal human cell models
Kisheon Alexander1, [email protected], Yueting Wu1,
Jacob Kaiser2, Yogesh Kulkarni1, David Klinke II1,2. (1) Department
of Chemical Engineering, West Virginia University, Morgantown,
West Virginia 26505, United States (2) Department of Microbiology,
Immunology and Cell Biology, West Virginia University, Morgantown, West
Virginia 26505, United States
Tumor-escape from the cytotoxic action of the immune system
is an emergent hallmark of cancer. Although our understanding
of how tumors suppress immunosurveillance remains unclear,
human cancer develops resistance to targeted therapies after
initial success. More comprehensive targeted therapies may be
developed on understanding the cross-talk between cancer cells
and immune cells. This study aims to identify secreted biochemical
signals secreted by malignant cells to skew immune response.
Three cell lines were investigated - two breast cancer cell lines
(BT474 and SKBR3) and a normal human mammary epithelial
cell line (184A1). A proteomics workflow that includes 2DE,
MALDI-TOF mass spectrometry, and PMF were used to analyze
the proteome. Collectively, the results suggest commonalities and
differences among the cell lines. Proteins associated with exosomes
were identified in the secretome and SEM confirmed exosome
presence. Differences in secreted proteome in malignant cell lines
were also confirmed by western blot. Future studies focus on
elucidating the role of tumor-derived secreted protein on immune
cell function. In summary, proteomics provides a less biased
approach to understand the biochemical signals that malignant
cells use to escape immunity and better understanding of cell-to
cell communication in breast cancer.
BIOT 278
Successful scale-up to 500L pilot-scale bioreactor for mass
production of exopolysaccharides by mycelial cells of
schizophyllum commune: The importance of oxygen mass
transfer rate and carbon/nitrogen ratio of production medium
Yong-Man Jang1, [email protected], yong-seob Jeong2, Gie-Taek
Chun1. (1) College of Biomedical Science, Kangwon National University,
Chuncheon-si, Gangwon-do 200-701, Republic of Korea (2) Faculty of
Biotechnology, Chonbuk National University, Jeonju-si, Jeonbuk 561-756,
Republic of Korea
Bioprocess scale-up from laboratory to pilot-scale was successfully
carried out for mass production of exopolysaccharide(EPS), an
active ingredient for skin moisturizer produced by mycelial cells of
Schizophyllum commune in submerged cultures. For development
110
of a pilot-scaled bioprocess, the effects of various environmental
factors on EPS production and cell growth were investigated,
firstly in both 250ml shake-flask and 5L bioreactor cultures. C/N
ratio was observed to have the most significant influences on the
EPS biosynthetic capability of the high-yielding mutants. With
the increment of the C/N ratio, the EPS production was also
enhanced, and vice versa. In the shake-flask fermentations, 15g/L
of EPS was produced at the C/N ratio of 27:1, which had been
statistically determined through response surface method (RSM).
Unfortunately, when the parallel fermentations were performed
in 5 liter bioreactors under the identical culture conditions to the
shake flasks, almost 40% reduction in the EPS productivity was
demonstrated, plausibly due to the apparent differences of oxygen
mass transfer coefficient (kLa) between the two culture systems.
Therefore, various agitation conditions (150, 200, 250rpm) and
C/N ratios were reexamined in the 5L bioreactor cultures, resulting
in almost similar level of EPS productivity at the agitation speed of
250 rpm and the C/N ratio of 40:1 to that from the previous shakeflask cultures. Notably, when dense filamentous morphologies
of the producing cells (to be transferred as active inoculums to
the final production fermenter) were induced by reducing shear
stress in the growth bioreactor cultures, more stable and higher
production of EPS was observed, reaching almost 20 g/L in the
final 5 liter production fermentations. By carefully applying all
the experimental results obtained from the bench-scale 5 liter
bioreactor cultures, it was possible to obtain almost similar level of
EPS productivity (18 g/L) in the 500 liter pilot-scaled fermentations.
BIOT 279
Application of scale-up criterion of constant volumetric oxygen
mass transfer coefficient (kLa) for successful production of
itaconic acid in a 50L pilot-scale fermentor by fungal cells of
Aspergillus terreus
Woo-Shik Shin1, Yong-Seob Jeong2, Sangyong Kim3, Dohoon Lee3,
Gie-Taek Chun1, [email protected]. (1) College of Biomedical
Science, Kangwon National University, Chuncheon-si, Gangwon-do 200701, Republic of Korea (2) Faculty of Biotechnology, Chonbuk National
University, Jeonju-si, Jeonbuk 561-756, Republic of Korea (3) Korea
Institute of Industrial Technology, Cheonan-si, Chungnam 331-825,
Republic of Korea
Scale-up criterion of constant oxygen mass transfer coefficient(kLa)
was applied for a successful production of itaconic acid(IA) in a
50L pilot-scale fermentor by the fungal cells of Aspergillus terreus.
Various operating conditions were examined to collect as many kLa
data as possible by adjusting stirring speed and aeration rate in 5L
and 50L fermentor systems. In the fermentations performed with
the 5L fermentor, the highest IA production was observed under
the operational conditions of 200rpm and 1.5vvm. Accordingly, we
intended to find out the parallel agitation and aeration rates in the
50L fermentor system, resulting in almost equivalent value of kLa
to that(0.02 sec-1) obtained from the 5L system. The conditions
of 180rpm and 0.5vvm turned out optimal for transferring almost
same volumetric amount of dissolved oxygen(DO) into the 50L
fermentaion broth as that observed in the 5L system, without
causing shear damages to the producing cells due to excessive
agitation. Consequently, almost identical fermentation physiology
was revealed in the 5L and 50L cultures performed under those
respective conditions, as expressed in terms of maximum IA
production(Pf) (51.2 vs. 52.7 g/L), volumetric(Qp) (0.533 vs.
0.548 g/L/hr) and specific(qp) (0.045 vs. 0.046 g/g DCW/hr) IA
production rates, and IA production yield(Yp/s) (0.711 vs. 0.715
g/g). Notably, the difference of specific growth rate(μ) between the
two cultures was observed to be negligible(0.029 hr-1 vs. 0.031
hr-1). This result was very impressive, considering the fact that
μ normally has a great influence on qp, especially in secondary
metabolites production.
BIOT 280
Statistical optimization of medium and fermentation
conditionsfor Xylanase production by recombinant Pichia
pastoris (xylanaseC + ρPICZαA)
Min-Yuan Zhang1, Taeyoung Ryoo2, Gie-Taek Chun3, Yong-Seob
Jeong1, [email protected]. (1) Department of Food science &
Technology, Chonbuk National University, Jeonju-si, Jeollabuk-do 561756, Republic of Korea (2) Research Center for Industrial Development of
Biofood Materials, Jeonju-si, Jeollabuk-do 561-756, Republic of Korea (3)
College of Biomedical Science, Kangwon National University, Chuncheonsi, Gangwon-do 200-701, Republic of Korea
For mass production of xylanase, statistical experimental methods
were applied to optimize production-medium composition using a
recombinant Pichia pastoris strain(xylanaseC + ρPICZα). Firstly,
medium components supposed to have positive effects on xylanase
biosynthesis were selected by preliminary experiments; one factor
at a time method. Then, the factors showing significant effects
were carefully chosen by Plackett-Burman design. Subsequently,
fractional factorial design (FFD) was used to investigate the
interactive effects of the selected medium components on xylanase
productivity. Based on the FFD results, steepest ascent method
(SAM) and response surface methodology (RSM) were employed
to finally determine the optimal concentrations of each component
for maximal production of xylanase. At the optimized medium
composition, 3051.7 mU/mL of xylanase was obtained, almost
three times higher amount compared to that from the initial
medium condition. In addition, environmental fermentation
conditions were intensively examined using the optimized
production medium in a laboratory bioreactor.
BIOT 281
Application of 24 deepwell plate fed-batch cultures for high
throughput screening in stable cell line development
Shunsuke Ohira1, [email protected], Yasuhiro Takagi2, Yun Seung
Kyung1, Tsuyoshi Nakamura2, Akira Egashira2, Marie Zhu1, Masami
Yokota2. (1) Department of Process Sciences, Agensys, Santa Monica, CA
90404, United States (2) Bio-Lead Project, Astellas Pharma Inc., Tsukuba,
Ibaraki, Japan
Because of the advantage of allowing screening of clones in
suspension culture at the earlier stage, many companies recently
introduced 24 deepwell plate batch culture for stable cell line
development, especially for screening and expanding desirable
clones. Screening of the clones in suspension culture is more
predictive than those in static culture. However, screening of the
clones in batch mode ignores the response of clones to the nutrient
feeding applied in fed-batch production, therefore, may lead to loss
of the best potential clones. In this work, we investigated a feasibility
of 24 deepwell culture in a fed-batch mode for early screening of
clones in the GS-CHO cell line development. We developed the
feeding strategy for the 24 deepwell plate fed-batch cultures and
compared the cell growth and antibody production in 24 deepwell
plates to those in shake flasks. The results show similar cell growth
and antibody production in both 24 deepwell plates and shake
flasks. Our data demonstrates that application of the 24 deepwell
fed-batch cultures provides a better high-throughput screening for
cell line development in terms of increasing screening throughput
and reducing shaker space and medium volume required by shake
flasks.
BIOT 282
Novel endogenous molecular reporters from engineered TetR
regulatory protein
Christopher Frei1, [email protected], Joseph Gredell1, Shuang-Yan
Tang2, Patrick Cirino1. (1) Department of Chemical and Biomolecular
Engineering, University of Houston, Houston, Texas 77004, United States
(2) Department of Chemical and Biomolecular Engineering, Pennsylvania
State University, University Park, Pa 16802, United States
Combinatorial design approaches to improve enzymatic or
microbial production of a metabolite require high-throughput
and selective screening systems. Regulatory proteins controlled
by “effector” molecules naturally couple molecular recognition to
changes in gene expression, providing a platform for linking in vivo
molecular synthesis to a readily detectable phenotype (e.g. GFP
expression). We are developing customized molecular reporters
by engineering regulatory protein effector recognition. In addition
to enabling high throughput screening, customized regulatory
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BIOT 277
BIOT 283
Effective phase III/commercial monoclonal antiboody
producing CHO cell line selection
Dacia Brooks, [email protected], Meg Tung, Martin Gawlitzek.Late
Stage Cell Culture, Genentech, South San Francisco, California 94080,
United States
When an improved cell line is needed for Phase III/commercial
development, the goal is to select a cell line with productivity
that meets the projected commercial product demand and with
similar product quality profiles to early clinical material. A series
of experiments were conducted over a period of six months
using a combination of high-throughput cell culture techniques
and 2 L bioreactors on eight candidate improved clones, while
simultaneously performing initial process development and laying
a solid foundation for future process optimization. Stability of the
cell lines was also monitored. As a result, we were able select a
cell line that met the titer and product quality requirements. Data
will be presented demonstrating the efficiency of our approach
for identifying the appropriate cell line for Phase III/commercial
process development.
BIOT 284
Fast neutron and alpha particle mutagenesis for the
exploration of gene function and biofuel production
enhancement in yeast
Jeremy Pearson1, [email protected], Oliver Jan1, George Miller2,
Nancy Da Silva1, Mikael Nilsson1. (1) Department of Chemical
Engineering, University of California Irvine, United States (2) Department
of Chemistry, University of California Irvine, United States
Mutagenesis of microorganisms has successfully been employed to
increase production of desired molecules and elucidate unknown
gene function. Fast neutron and ion beam mutagenesis are examples
which utilize high linear energy transfer (LET) particles. High
LET particles are noted for their ability to create clustered lesions
and double strand breaks in DNA resulting in strong mutational
effects. These increase the probability for genetic knockouts which
are useful for identifying gene function and dramatically altering
metabolic pathways. Here we demonstrate a method for creating
112
high LET particles in situ utilizing the 10B(n,α)7Li reaction which
is currently studied in boron neutron therapy cancer research, and
apply it towards mutagenesis of biofuel producing organisms. Yeast
is irradiated in the presence of boron to create mutants. Enhanced
fatty acid producing strains are screened to quantify increase in
fatty acid production and determine alterations to the genome
which may be associated with this increased production
BIOT 285
Production of ethanol from Saccharomyces pastoranius on
biomass sugars using a two-stage fermentation process
Yogender Kumar Gowtham1, [email protected], Kristen
Miller2, Michael Henson2, Sarah Harcum1. (1) Department of
Bioengineering, Clemson University, Clemson, SC 29631, United States (2)
Department of Biological Sciences, Clemson University, Clemson, South
Carolina 29631, United States
Saccharomyces cerevisiae has been widely used in industry for
the production of bioethanol. Saccharomyces pastoranius, which
is related to S. cerevisiae, is also known to produce similar levels
of bioethanol, but is widely used to make lager. S. pastorianus has
a wider temperature tolerance and potentially greater tolerance
to inhibitors found in biomass sugar hydrolysates. A two-stage
fermentation process was used to produce bioethanol from
switchgrass hydrolysates. The first stage focuses on consumption of
glucose, and simultaneous production of bioethanol. To assist xylose
consumption, xylose isomerase was added to the fermentation to
convert xylose. In the presence of xylose isomerase, S. pastoranius
showed significantly improved growth with xylose isomerase;
however, the observed increase in growth was not due to xylose
conversion (only 10%). The unconverted xylose from the first stage
was fed to a second stage fermentation. The two-stage fermentation
process significantly improved the overall bioethanol productivity.
BIOT 286 – Withdrawn
BIOT 287
High-throughput screening of HIC media in PreDictor plates
for capturing recombinant green fluorescent protein from E.
coli
Carina Engstrand, [email protected], Charlotte Brink.
Chemistry, GE Healthcare, Uppsala, Uppsala 75184, Sweden
HIC is a powerful purification technique where the type and density
of the ligand, pH and salt of binding conditions, temperature and
the nature of the target protein are highly significant parameters
in finding and fine tuning selectivity. High throughput process
development in plates is a valuable tool for rapid evaluation of
operating conditions for a HIC step.
This work will present a high throughput screening study for the
development of a capture step for recombinant Green Fluorescent
Protein (rGFP) expressed in E. coli. Parallel screening of HIC
media and conditions in 96-well plates facilitated the selection of
the most promising HIC media and also show how salt type and salt
concentration conditions could be tailored to successfully capture
rGFP. Results showed that both binding and elution conditions
could be fine-tuned to obtain high protein purity. This approach
allows selection of HIC media and elution profiles to be predicted
in a fast and effective way, saving both time and sample compared
with traditional column screening.
BIOT 288
Mechanisms of improved n-butanol tolerance and production
revealed by comparative proteomic and genomic analyses of a
mutant strain of Clostridium
Jingbo Zhao, [email protected], Shang-Tian Yang.Department
of Chemical & Biomolecular Engineering, The Ohio State University,
Columbus, OH 43210, United States
n-Butanol is an important industrial chemical and next-generation
biofuel. Bioprocessing of renewable resource to n-butanol is
environmentally friendly, but hampered by the low n-butanol
titer and consequently high recovery cost. A mutant strain of
Clostridium achieved by adaptive evolutionary engineering could
produce 21 g/L and 28 g/L n-butanol in free and immobilized cell
fermentations, respectively. The mutant and its parent strain were
used to reveal the mechanisms of improved n-butanol tolerance
and production by comparative proteomic and genomic analyses.
The proteomic profiles of both strains were analyzed by 2-D gel
electrophoresis. Proteins with dramatic expression difference
were identified and classified. Complete genomes of both strains
were sequenced using the high-throughput Illumina sequencing
technology and analyzed. SNP and Indel analyses revealed 7
variations contributing to the characteristics of the mutant strain.
The results can be used in further strain improvement via inverse
metabolic engineering.
BIOT 289
BDSF quorum sensing molecule is synthesized by a
bifunctional crotonase homologue that has both dehydratase
and thioesterase activities
Hongkai Bi, [email protected] of Illinois, United
States
Various gram-negative pathogenic bacteria use fatty acids of
medium chain length collectively called Diffusible Signal Factors
(DSFs) as quorum-sensing molecules to regulate virulence and
biofilm formation in response to the local cell concentration.
The pathway of DSF biosynthesis was unclear since these fatty
acids contain an unusual cis-2 double bond. We report that
cis-2-dodecenoic acid (called BDSF), the DSF of the human
pathogen Burkholderia cenocepacia, is synthesized from the
3-hydroxydodecanoyl-acyl carrier protein intermediate of fatty
acid synthesis by intercession of a protein, Bcam0581, that is
a homologue of the classical b-oxidation enzyme crotonase.
Bcam0581 has both dehydratase activity and thioesterase activity.
Both activities are abolished by mutagenesis of active site residues
identified by alignment with rat mitochondrial crotonase. Although
dehydratase/hydratase activity is the classical crotonase reaction
and two instances of thioesterases that are crotonase superfamily
members have been reported, this is the first such enzyme that has
both activities. The chemical and biological advantages of BDSF
synthesis such a bifunctional enzyme will be discussed.
BIOT 290
Butanol fermentation integrated with adsorption for in situ
recovery
Fangfang Liu, [email protected], Chuang Xue, ShangTian Yang.
Chemical and Biomolecular Engineering, The Ohio State University,
Columbus, OH 43210, United States
Biobutanol is increasingly attracting attention as an alternative to
traditional petroleum-derived fossil fuels. Compared to ethanol,
butanol has several advantages including high energy density,
low water absorption and easy application to the existing gasoline
engine. However, butanol fermentation has several obstacles:
the low butanol concentration in the fermentation broth due
to the toxicity of butanol to organisms that produce it, and the
lack of an energy-efficient method of recovering butanol from
the fermentation broth. Many studies tried to recover butanol
using in-situ recovery technologies, including gas stripping,
extraction, adsorption, etc. This research focused on butanol
fermentation with in-situ adsorption to alleviate butanol toxicity
and increase butanol production. Various adsorbents, including
113
Tuesday Afternoon
proteins are useful tools in metabolic engineering applications.
Our success in using AraC variants as endogenous reporters has
motivated similar efforts with other regulatory systems. The TetR
repressor is well suited for detecting polycylic natural products.
This poster describes our progress in optimizing and screening
TetR libraries to design novel reporters for various polyketides.
BIOT 291
Engineering bacteria for improved hydrocarbon efflux
Jee Loon Foo, Matthew Wook Chang, Susanna Su Jan Leong,
[email protected] of Chemical and Biomedical Engineering,
Nanyang Technological University, Singapore, Singapore
Escherichia coli is the production host used for the production of
many biofuels. However, many organic solvents are toxic to bacteria
and the tolerance of bacteria to these solvents has been found to
involve exportation of the molecules out of the cells through efflux
pump systems. In the context of biofuel production, these efflux
pumps could be employed to confer greater solvent tolerance of
the production host as well as promote secretion of the produced
biofuel out into the external medium, thus increase the efficiency
and reduce cost of biofuel production.
Of the few efflux pump systems identified in E. coli, the AcrABTolC is probably the best characterised. In order to facilitate the
use of the AcrAB-TolC system for higher efflux efficiency, we
have performed directed evolution on the AcrB inner membrane
transporter for increased efflux of organic molecules, in this
case, n-octane. Mutant libraries of AcrB were generated through
random mutagenesis and variants with accelerated extrusion of
intracellular n-octane were isolated after enrichment screening
based on the n-octane tolerance of E. coli transformed with the
mutant genes. The engineered AcrB mutants could be utilized
in E. coli for more efficient biofuel production. Additionally, the
information gathered from the mutants would enable us to further
our understanding of the pump and aid rational design of the efflux
system in the future.
BIOT 292
Mathematical model for kinetic predictability of biopolymer
breaking enzyme system for oil wells
Debayan Ghosh1, [email protected], Bodhisattwa Chaudhuri2.
(1) Department of Research, Epygen Labs FZ LLC, Dubai, Dubai 485018,
United Arab Emirates (2) Department of Pharmaceutical Sciences and
Institute of Material Sciences, University of Connecticut, Storrs, CT 06269,
United States
For a horizontal well, an environmentally cleaner substitute to Oil
based mud systems, the Water based mud Biopolymer-Carbonate
114
drill-in fluid deposits an impermeable Biopolymer filter cake on
the borehole wall. This Biopolymer barrier must be completely
broken to maximize oil production rates. Specially designed
Enzyme proteins can substitute harsh breaking chemicals and can
be designed for complete dissociation of Biopolymer based mud
damage. Since most enzymes have a tendency to get completely
denatured at high temperatures and extreme pH conditions in
wellbores, the objective of this study was to establish a mathematical
model predicting the effectiveness of the new generation enzyme
system to clean freshly drilled wells at high temperature and
variable pH environment. This study includes modeling of the
Kinetic aspect of enzyme action, predicting how effectively the
enzyme is converting Biopolymer to water soluble reducing sugar,
rendering the filter-cake easily removable within a known span of
time.
BIOT 293
High-throughput HepG2-based microarrays for studying 3D
cell culture
Luciana Meli1, [email protected], Eric T. Jordan2, Robert J. Linhardt1,
Jonathan S. Dordick1. (1) Department of Chemical and Biological
Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180,
United States (2) Department of Biology, Rensselaer Polytechnic Institute,
Troy, New York 12180, United States
We have developed a 3D cellular microarray platform for the highthroughput analysis of growth, cytotoxicity, and protein expression
profile of a human hepatocellular carcinoma cell line, HepG2, in
alginate, and compared these results to 2D and 3D environments
at the microwell plate scale. The antiproliferative effects of four
drugs, tamoxifen, 5-fluorouracil, doxorubicin, and amitriptyline,
were studied as a function of seeding density in the three culture
platforms. The chemosensitivity of HepG2 cells to 5-fluorouracil
and doxorubicin decreased substantially with increasing cell
number in all surfaces/matrices, but little change in the IC50
values was observed between the different culture platforms when
normalizing to the seeding density. Additionally, we developed
a chip-based in-cell immunofluorescence assay that provided
quantitative data of the levels of specific target proteins involved
in proliferation, cell-cycle regulation, adhesion, angiogenesis,
and drug metabolism, and compared the expression profiles to
analogous results in 2D environments.
BIOT 294
Expression of modular collagen and variants in Saccharomyces
cerevisiae
the distinguishing characteristic of M. sp. DH is that the culture
still could thrivingly grow on methane after repeated growth on
carbohydrates.
Sam Wei Polly Chan, [email protected], Szu-Wen Wang, Nancy A
BIOT 296
University of California, Irvine, Irvine, CA 92697, United States
Fumaric acid production by Rhizopus oryzae immobilized
on static fibrous bed bioreactor with in situ recovery by ion
exchange
Da Silva.Department of Chemical Engineering and Materials Science,
We have created a de novo full-length modular collagen gene
that enables fabrication of specific mutants with high potential
in regenerative medicine, tissue engineering and drug delivery.
Although native collagen is typically extracted from animals, safety
concerns and variability limitations of animal protein drive the
development of recombinant expression systems such as yeast. In
developing our modular collagen gene, we overcame the challenges
of mishybridization during PCR and site-directed mutagenesis,
and can mutate at defined locations, combinations and frequencies.
To face the challenge of expressing thermally stable collagen with
the desired hydroxylation percentage, we developed different
integrant and plasmid systems that co-express the two subunits
of human prolyl hydroxylase and collagen at different gene ratios
and expression levels. We report the different hydroxylation
abilities of these systems and demonstrate that the resulting fulllength collagen and variant proteins can be successfully expressed,
purified, and characterized.
BIOT 295
Microbacterium sp. DH, a novel aerobic, facultative and
methane-oxidizing bacterium isolated from aged refuse
Kun Zhang, [email protected], Shang-Tian Yang.Department of
Chemical and Biomolecular Engineering, Ohio State University, Columbus,
OH 43210, United States
Production of fumaric acid (FA) by fungal fermentation from
renewable resources has received much attention due to the
increased production cost of petroleum-based FA. However,
current fermentation process suffers from low productivity due to
difficulty in controlling fungal morphology and product inhibition.
In this study, an integrated system coupling fermentation and ion
exchange for in situ recovery of FA produced by Rhizopus oryzae
was investigated. A static fibrous bed bioreactor (SFBB) was used
for immobilizing fungal mycelia and controlling their morphology
by the formation of a biofilm. IRA900, an ion-exchange resin with
a high FA adsorption capacity at pH 5 and high selectivity, was
used for in situ FA recovery. With the ion-exchange column, FA
produced in the SFBB was recovered simultaneously to alleviate
inhibition, sustain cell viability and enhance FA productivity. Also, pH was
maintained in the optimal range without adding any base, avoiding excessive
CaCO3 for pH control.
Tiantao Zhao1,2, [email protected], Lijie Zhang1,2, ShangTian
BIOT 297
Chongqing, Chongqing 400050, China
Sensitivity of null and recombinant Escherichia coli HMS174
to high agitation rates and low inoculation levels: A
cautionary tale for seed train design
Yang1. (1) Dept. of Chemical & Biomolecular Engineering, The Ohio
State University, Columbus, Ohio 43202, United States (2) Dept. of
Chemistry and Chemical Engineering, Chongqing University of Technology,
Facultative methanotrophs reported recently could only utilize a
few of C2, C3 and C4 compounds, which limits the application
of methane oxidization in the engineering of greenhouse gas
reduction due to the difficulty of propagation. A novel strain,
Microbacterium sp. DH isolated from aged refuse, can utilize
methane as well as multi-carbon compounds including organic
acids and carbohydrates. Characteristics of the growth of M. sp.
DH were studied with different C6 saccharides as carbon and
energy sources, which included glucose, mannose, sucrose, lactose,
raffinose and starch, and the maximum specific cell growth rates
(μmax) were derived using Boltzmann simulation and interpolation
methods. The values of μmax ranged from 0.068 h-1 to 0.085 h-1,
which indicated that C6 saccharides could be utilized by M. sp.
DH as well as methane (μmax = 0.079 h-1, R2= 0.9951). Moreover,
Kathrine Allikian, Varnika Roy, [email protected], Mark Berge.
Department of Process Cell Culture and Fermentation, MedImmune,
Gaithesburg, Maryland 20878, United States
Establishment of a scalable seed train is an integral part of any
fermentation process development initiative. Multi-stage seed
trains use a combination of shake flasks and bioreactors to expand
the culture biomass to reach proper inoculum densities for the
production fermentor. Generally, for bacterial systems, there is
little difference between the growth in shake flasks and bioreactors,
but a recent study at MedImmune has shown otherwise. As part
of MedImmune’s development of a fermentation process using
recombinant Escherichia coli HMS174, we grew the second stage of
a two-stage seed train in a bioreactor under high agitation with an
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Tuesday Afternoon
activated carbon, resin L-493 and SD-2 were evaluated for their
butanol adsorption capacity. These adsorbents were then tested
in the integrated adsorption-fermentation, which greatly reduced
butanol inhibition and enhanced butanol yield, productivity and
the final butanol titer.
BIOT 298
Direct conversion of cellulose to cellobionate using an
engineered Neurospora crassa without cellulase addition
BIOT 299
monoclonal antibodies and viral vaccines.
Evolving platform process for producing novel monoclonal
antibodies and antibody products
BIOT 302
Bridget Leslie1, [email protected], Nick Alden1, Loray Paul1,
Rochelle Shapland1, Pratik Jaluria1, Adam Lucka2. (1) Department of
Upstream Development, Alexion, Cheshire, CT 06410, United States (2)
Department of Protein Characterization, Alexion, Cheshire, CT 06410,
United States
Monoclonal antibody therapies and antibody-based products are
characterized, evaluated for efficacy and scaled up for pre-clinical
production early in development. Cell banking, seed train and
production bioreactor are three major upstream processes that
need to be developed to ensure that consistent and homogeneous
product can be achieved. A platform cell culture production process
provides a useful starting point for generating consistent preclinical
material for characterization studies and for evaluating critical
process parameters. We developed a CHO-specific platform process
that was originally designed and refined for a series of monoclonal
antibodies and applied it to an antibody fragment. Concurrent
with our evaluation of critical process steps, we screened cell lines
to select a single cell line for clinical manufacturing and introduced
new elements of our platform process including optimized feed
formulations. Improvements in robustness and productivity have
been realized based on our approach.
Amanda Hildebrand1, [email protected], Julia Fan1, Weihua
Wu1, Takao Kasuga2. (1) Department of Biological and Agricultural
Engineering, University of California, Davis, Davis, CA, United States (2)
Department of Plant Pathology, University of California, Davis, Davis, CA,
United States
In this study, we investigate the production of cellobionate from
cellulose by a genetically modified fungus, Neurospora crassa.
Cellulose was diverted to cellobionate by knocking down multiple
copies of the beta-glucosidase gene. The accumulated cellobiose
was converted to cellobionate by another enzyme, cellobiose
dehydrogenase, produced by N. crassa. In this way, cellobionate is
produced without requiring the addition of exogenous cellulase.
Although 50% of cellulose was diverted to cellobiose production,
the concentration of cellobionate produced was quite low. We report
the improvement of the cellobionate yield through the addition of
a catalytic amount of redox mediator, with in-situ regeneration of
the redox mediator by laccase (Zhao and van der Donk, 2003), and
the optimization of the culture conditions.
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BIOT 300 – Withdrawn
BIOT 301
Efficient evaluation of alternative technologies for
biomanufacturing
Charles Siletti, [email protected], Demetri Petrides, dpetrides@
intelligen.com.Intelligen, Inc., Scotch Plains, NJ 07076, United States
The successful scale up and commercialization of biopharmaceuticals
is a challenging task that requires collaboration of professionals
from many disciplines. Process simulators and other computer
aids can facilitate this task by assisting scientists and engineers to
efficiently answer the following and other related questions: What
is the impact of product titer increase on the capacity load of the
downstream section, the overall throughput of a plant, and the cost
of goods? What is the impact of single-use systems on the demand
for utilities, the environment, and the cost of goods? What is the
range of variability that a process can accommodate if it operates
under a tight cycle time? Our experience in addressing the above
questions will be presented using industrial examples in which
we evaluated alternative technologies for producing therapeutic
Action of Savinase and papain treatments on the properties of
nylon 6,6 fibers
Ping Wang, li Cui, [email protected], Jiugang Gang, Qiang Wang,
Xuerong Fan.Key Laboratory of Science and Technology of Eco-Textile,
Ministry of Education, Jiangnan University, Wuxi, Jiangsu 214122, China
In this paper, the proteases of Savinase and papain were applied in
the processing of nylon 6,6 fabrics, respectively, aiming at improving
the fiber properties by changing the enzyme concentrations,
incubating time and so on. The properties of the polyamide fibers,
before and after protease treatments were detected, including
dyeability, weight loss, surface morphology and thermodynamic
performance. The result indicated that Savinase could partially
hydrolyze the amide- bonds during the enzymatic processing,
resulting in a slight increase of dyebath exhaustion. However,
papain treatment did less impact on the properties of the nylon
6,6 fabric. SEM implied that Savinase could partially degrade the
surface of nylon 6,6 fibers, resulting a slight alteration of the fiber
surface. The data of DSC showed that Savinase treatment could
slightly increase the glass transition temperature of the polyamide
fiber, but the other thermodynamic properties had no noticeable
changes. The experiments indicated that protease treatment just
hydrolyzed the amide- bonds in the outmost surface of nylon 6,6
fibers, and the integrality of fiber interior scarcely changed. The
degree of polymerization, crystallinity and orientation for the
protease-treated polyamide fiber were also similar to that of the
untreated one.
BIOT 303
industrial relevant carbon and nitrogen sources, which showed
consistent performance for over 20 consecutive batches with high
titer, yield and productivity. Further optimization of the media
composition was performed to reduce the overall media cost for
economical production of n-butanol in ABE fermentation. The
results confirmed that JB200 can be used to produce n-butanol
economically in industry to meet the rapidly increasing market
demand.
BIOT 304
Use of thin stillage from dry mill ethanol plant for producing
omega-3 fatty acid by microalgae
Yi Liang, [email protected], Zhiyou Wen.Department of Food Science
and Human Nutrition, Iowa State University, Ames, IA 50010, United
States
The use of ethanol as transportation fuel has stimulated its
production in the past years. This has led to the accumulation of
large amounts of byproducts. In US, specifically, the byproducts
from dry mill ethanol plants are the residues after distillation of
ethanol, namely whole stillage, which are usually supplemented
into ruminant feed. The purpose of this study is to investigate
the potential use of thin stillage (a fraction of whole stillage), as
a cheap nutrient source, to grow an omega-3 fatty acid producing
microalgae species, Pythium irregulare. We tested the effects of
thin stillage concentrations on the cell growth and omega-3 fatty
acid productivity. The result showed that, at 50% thin stillage
concentration, 120 mg/L EPA can be produced by Pythium
irregulare. The total nitrogen, total phosphorus, and COD
removal efficiency in the culture medium were 53%, 44% and 75%,
respectively. This nutrient-depleted water can be recycled to corn ethanol
production facilities, which can mitigate the high production cost from water
used.
Long-term stability and butanol production in repeated batch
fermentations of Clostridia acetobutylicum JB200
BIOT 305
Wenyan Jiang, [email protected], Jingbo Zhao, Shang-Tian Yang.
Protein endocytosis and degradation: Impact on secreted
protein titers in yeast
Department of Chemical and Biomolecular Engineering, The Ohio State
University, Columbus, OH 43210, United States
Butanol is a widely used chemical and a promising biofuel.
Clostridium acetobutylicum JB200 is an adaptive mutant with
high butanol tolerance and can produce up to 29 g/L butanol from
glucose in a fibrous-bed bioreactor (FBB). As one of the best strains
ever reported for acetone-butanol-ethanol (ABE) fermentation, it
is a favorable host for industrial biobutanol production. However,
its long-term performance in fermentation with industrial media
remains unknown. In this study, the long-term stability and
fermentation performance of JB200 were tested with media using
Keith EJ Tyo1, [email protected], Zihe Liu2, Dina Petranovic2,
Jens Nielsen2. (1) Department of Chemical and Biological Engineering,
Northwestern University, Evanston, IL 60201, United States (2)
Department of Chemical and Biological Engineering, Chalmers University
of Technology, Evanston, IL 60201, United States
Recombinant protein production is an important activity in the
production of drugs, fuels, and chemicals. Secreting protein into
minimal media simplifies downstream purification, therefore
reducing cost of manufacture. To date, considerable effort has been
placed on increasing protein titers through improving recombinant
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Tuesday Afternoon
inoculation level of 0.1% from a mid-exponential phase shake flask
culture using a complex medium. Under these conditions, we have
observed no growth at three different fermentor scales (1-L, 15-L
and 100-L), whereas parallel shake flask cultures under the same
conditions grow at acceptable rates. Upon further investigation, we
have found that E. coli HMS174 is sensitive to a combination of
agitation, cell density (inoculation level), and, to a lesser degree,
antifoam concentration. At the 1-L scale, agitation rates of < 400
rpm have supported growth with a 0.1% inoculation level. Further
investigation has shown that growth is supported at agitation rates
of up to 1200 rpm but with a 1.25% inoculation level. Additionally
we tested the growth of null and recombinant versions of both E.
coli BL21(DE3) and DH5a cells under a subset of the experimental
conditions. We found that BL21(DE3) grows well at high agitation
rates and low inoculation levels, while DH5a is more sensitive
to agitation at low inoculation levels. Based on these small-scale
findings, our final production seed train for the recombinant E.
coli HMS174 strain incorporates considerable modifications to
agitation rate, inoculation level, and antifoam concentration to
achieve an acceptable growth profile.
BIOT 306
Speed meets quality: Challenges in developing a cell culture
process for a recombinant protein
Rochelle M. Shapland1, [email protected], Anne Kantardjieff1,
John Facenda1, Adam Lucka2, Natalia Corleone1, Pratik Jaluria1. (1)
Department of Upstream Development, Alexion, Cheshire, CT 06410,
United States (2) Department of Protein Characterization, Alexion,
Cheshire, CT 06410, United States
Rapid advancement of molecules into clinical studies necessitates
establishment of a robust, reliable cell culture platform process.
We have developed a platform process using multiple CHO cell
lines producing monoclonal antibodies. The platform process we
developed was applied to a CHO cell line developed externally by
a CMO, producing a fusion protein. We evaluated their process
with several requirements in mind, paramount being preservation
of product quality attributes. Additional requirements included:
changing cell lines from CMO-generated to in-house generated,
use of chemically defined medium and feeds and improved
manufacturability. Our evaluation indicated subtle differences in
product quality, specifically glycosylation, observed between our
cell lines and the CMO-generated cell line. However, other factors
contributing to product quality included: bioreactor scale, media
composition, feeding schedule, and harvest criterion. Ultimately,
we successfully developed a process using an in-house generated
cell line exhibiting comparable product quality attributes.
BIOT 307
Development of a scale-down model using the Micro24
micro-bioreactor system
Nicholas Alden, [email protected], Anne Kantardjieff, Pratik Jaluria.
Department of Upstream Development, Alexion Pharmaceuticals, Cheshire,
CT 06410, United States
A predictive small-scale model is a crucial tool for cell line selection
118
and process development. Commonly used models, such as well
plates and shake flasks, are often uncontrolled systems, which may
be less representative of controlled bioreactors. To this end, we
have developed a method for the operation of the Micro24 microbioreactor system which integrates pH, temperature and dissolved
oxygen control while maintaining a low volume. Success criteria
were defined as ease-of-use and comparability to other scales.
Method development included the optimization of inoculation
strategies, selection of appropriate gas mixtures and implementation
of a manual base addition strategy. Our results showed that, by
incorporating these changes, we were able to improve maximum
viable cell density by greater than 40% and the culture was extended
by approximately five days. Results were compared to various
scales and were found to be more comparable to larger scales (45L
and 15L stainless steel bioreactors).
BIOT 308
Direct conversion of glucose into lactate-based polyesters
using engineered Corynebacterium glutamicum as a whole cell
catalyst
Yuyang Song1, [email protected], Ken’ichiro Matsumoto1, Miwa
Yamada1, Aoi Gohda1, Christopher J Brigham2, Anthony J Sinskey2, Seiichi
Taguchi1. (1) Graduate School of Engineering, Hokkaido University, Sapporo,
Hokkaido 060-8628, Japan (2) Department of Biology, Massachusetts Institute
of Technology, Cambridge, Massachusetts 02139, United States
Lactate(LA)-based polyesters are promising bio-based materials
as alternatives to petroleum-based plastics. We have established an
engineered Escherichia coli system, which directly convert biomass
into LA-based polyesters. An LA-polymerizing enzyme (LPE)
plays a central role in the intracellular polymerization of lactic acid.
In this study, we used an alternative approach for meeting practical
use requirements in food-grade and medical-related applications.
We transferred the LA-based polyester biosynthesis machinery
from prototype E. coli system to the industrially significant and
safe organism Corynebacterium glutamicum, which does not
produce any toxic compounds (i.e. endotoxin).
The genes encoding the enzymes involved in monomer supplying
(lactyl-CoA and 3-hydroxybutyryl-CoA) and LPE were introduced
into C. glutamicum and the function of the individual enzymes was
confirmed. The GC/MS and NMR analyses of extracted polymers
revealed that the engineered C. glutamicum produced LA-based
polyesters with extremely high LA fractions of which were hardly
produced by the previous E. coli system.
BIOT 309
macromolecular crowding affect molecular mechanisms involved
in the operation and regulation of genetic circuits in living systems.
Macromolecule synthesis by DNA templated chemistry
Phillip J Milnes1, [email protected], Mireya L McKee2,
Jonathan Bath2, Eugen Stulz3, Andrew J Turberfield2, Rachel K O’Reilly1.
(1) Department of Chemistry, University of Warwick, Coventry, United
Kingdom (2) Department of Physics, University of Oxford, Oxford, United
Kingdom (3) Department of Chemistry, University of Southampton,
Southampton, United Kingdom
DNA adapters linked to reactants can direct small-molecule
chemical synthesis. If two reactants are linked to complementary
oligonucleotide tags then hybridization of the tags ties them closely
together, increasing the effective molarity with the potential to
greatly increase the reaction rate. Using this concept we present a
system which uses multi-step DNA templated chemistry to allow
the synthesis of sequence defined macromolecules. The Wittig
reaction was used to link ylide and aldehyde groups, concatenating
monomers attached to DNA adapter strands to form a growing
oligomeric chain. We have also been able to incorporate alkyne
side-chain functionality, thus allowing access a more diverse
products range via pre or post-functionalization.
References
M. L. McKee, P. J. Milnes, J. Bath, E. Stulz, A. J. Turberfield, R. K.
O’Reilly, Angew. Chem. Int. Ed. 2010 , 49, 7948.
P. J. Milnes, M. L. McKee, J. Bath, E. Stulz, A. J. Turberfield, R. K.
O’Reilly, In preparation.
BIOT 310
Control of mass transport and chemical reaction kinetics in
ultrasmall volumes
Charles P Collier, [email protected] for Nanophase
BIOT 311
Automated online sampling solution for improving cell culture
productivity
William Miller1, [email protected], Michael Biksacky1,
Chengbin Lin2, John Knighton3. (1) Flownamics, Inc., Madison, WI
53718, United States (2) Genzyme, Inc., Framingham, MA, United States
(3) Johnson & Johnson, Radnor, PA, United States
A SEG-FLOW™ automated online bioreactor sampling and
feed system (SEGFLOW) was evaluated for its impact on CHO
cell culture process performance in a bench-scale stirred tank
bioreactor. Real-time glucose analysis was performed by the
SEGFLOW using a biochemistry analyzer. Glucose setpoint
control was accomplished by the SEGFLOW through its feed
algorithm calculation and feed pump control scheme. A second
batch, using a daily manual sampling and bolus feed protocol,
was performed as a control. The SEGFLOW provided optimal
glucose feeding for the CHO cells, which allowed a greater carbon
conversion efficiency that reduced glucose uptake requirements,
increased peak and total viable cell concentrations and increased
protein yield. Reduced lactate production was also achieved, which
reduced base addition requirements and lowered media osmolality.
The SEGFLOW provided an innovative approach for improving
cell culture productivity through better process monitoring and
control, while increasing process knowledge and understanding
through real-time sample analysis.
BIOT 312
Materials Sciences, Oak Ridge National Laboratory, Oak Ridge,
TN 37831, United States
Robust optical in situ glucose sensor for biopharmaceutical
production
A better understanding of how confinement and reduced
dimensionality modulate chemical reactivity and reaction
dynamics will aid in the rational and systematic discovery of
functionality unique to nanoscale systems. This talk will describe
means for triggering chemical reactions for studying reaction
kinetics under extreme confinement with sub-millisecond
temporal resolution, including on-demand generation and fusion
of femtoliter-volume water-in-oil droplets, and triggering reactions
in femtoliter chambers microfabricated in poly(dimethylsiloxane)
(PDMS). We are also developing methods to vary confinement
and macromolecular crowding in ultrasmall, water-in-oil droplets
and chambers micromolded in PDMS as biomimetic reaction
vessels containing minimal synthetic gene circuits, in order to
better understand how confinement, reduced dimensionality and
Greg Emmerson, Sam Watts, George E Barringer, george.barringer@
stratophase.com.Stratophase Ltd, Romsey, United Kingdom
A robust, real time, solid state, in-situ optical sensor that tracks
glucose concentration has been developed and deployed in
fermentation and cell cultures. Successful implementation of the
real time, in-situ sensor for glucose monitoring will provide the
missing critical link in implementing real time feedback control
of bioreactors that is needed to improve product quality, yield, and
facility capacity. Specifications and statistical data will be discussed.
Applications in microbial and mammalian culture systems will be
shown and discussed.
119
Tuesday Afternoon
protein synthesis. In the present work, we unexpectedly identified
protein degradation as a significant process that decreases protein
titers in Saccharomyces cerevisiae. Experiments showing (1)
competitive endocytosis of insulin, bovine serum albumin (BSA),
and yeast extract protein and (2) growth on BSA as a sole carbon
source was used establish endocytosis as the mechanism of protein
loss. Transcriptomics showed decreased amino acid synthesis,
increased amino acid degradation/utilization, and metabolomics
showed increased concentrations of amino acids, indicating large
scale uptake of protein for carbon and energy. This work has
revealed an unanticipated mechanism for product loss that impact
many industrial and pharmaceutical bioprocesses.
Effects of steam explosion on digestibility and accessiblity on
loblolly pine in biofuel applications
Yuzhi Kang1, [email protected], Sang Beom Kim1, Prabuddha
Bansal1, Matthew Realff1, Andreas Bommarius1,2. (1) Department of
Chemical and Biomolecular Engineering, Georgia Institute of Technology,
Atlanta, GA 30332, United States (2) Department of Chemistry and
Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, United
their inhibition effects is needed. In this study, we investigated the
inhibition effects of various inhibitors derived from degradation
of carbohydrates and lignin on several solventogenic Clostridia
strains. Tolerance and fermentation kinetics of C. beijerinckii and
C. acetobutylicum exposed to these inhibitors at various levels were
determined, and the information was then used to optimize both
hydrolysis and detoxification processes. The improved hydrolysates
significantly increased butanol titer, productivity and yield in the
fermentation.
States
Steam explosion is one of the most effective pretreatment methods
in disturbing softwood structure and enhancing its accessibility. The
key operational parameters including temperature, residence time
and acid concentration have pronounced effects on crystallinity
(CrI) of the feedstock loblolly pine (LP). The CrI of pretreated LP
was determined from X-ray diffraction pattern with the method
of Least-squares and found to decrease as pretreatment severity
increase.
Three different substrates, Avicel, phosphoric acid swollen
cellulose (PASC), and SO2 steam exploded Loblolly pine (SELP),
were subjected to enzymatic hydrolysis and subsequent adsorption
study. All SELP samples with different pretreatment severities
were found more degradable by the Celluclast® enzyme system
than Avicel. Despite the complexity of both substrates and enzyme
mixtures, all SELPs adsorption isotherm can be fitted to a Langmuir
isotherm with R2 > 0.97. The highest binding capacity of SELP
was found to be ~185 µg/mg substrate at highest pretreatment
severity. Adsorption data was correlated with initial rates and a
linear relationship between initial rate and adsorption amount was
confirmed.
BIOT 314
Effects of lignocellulose derived inhibitors on butanol
fermentation of C. beijerinckii and C. acetobutylicum
Jie Dong1, [email protected], Congcong Lu2, Shang-Tian Yang1. (1)
Department of Chemical and Biomolecular Engineering, The Ohio State
University, Columbus, OH 43210, United States (2) The Dow Chemical
Company, Coatings Technology Center, DCM, United States
Ligncellulosic biomass is abundant and more economical for use in
fermentation to produce butanol, one promising biofuel. However,
ligncelluloses need to be hydrolyzed into monosaccharides
before fermentation. The hydrolysis process usually produces
some inhibitory compounds that could severely inhibit bacteria
growth and butanol production. Therefore, the inhibitors in the
hydrolysate must be reduced or removed by certain detoxification
processes before fermentation. To make the detoxification process
more efficient, a better understanding of these inhibitors and
120
BIOT 315- Withdrawn
BIOT 316
Modeling the entire process and finding the production and
cost limits
Charles Siletti, [email protected], Demetri Petrides, dpetrides@
intelligen.com.Intelligen, Inc., United States
Although the upstream and downstream processes are separated
from an organizational standpoint, optimizations in one area may
not necessarily lead to reduced costs or improved productivity in
the overall process. The results of a series of modeling studies of
the production of monoclonal antibodies and viral vaccines are
presented. The results indicate that there is an economic limit to
the value of increasing product titer in the bioreactor. The studies
also show that production bottlenecks may develop in support
areas, especially buffer preparation. The effectiveness of several
debottlenecking strategies, including in-line buffer dilution and
single-use containers, are presented.
Moreover, the size of the adipose in the cell is also smaller than
the one without nanoparticles. We, therefore, coated the TiO2
nanoparticles and try to prevent the damage of the uncoated TiO2.
As for our coating, we found higher conversion rate and also
larger size of adipose in the cells. These results indicated that the
predominant mechanism of damage by these nanoparticles will
cause different differentiation in pre-adipocyte cells and could be
prevent by our coating.
BIOT 318
Biosynthesis of the pyrolloquinoline alkaloid lymphostin in the
marine bacterium Sailinispora
Elisha N Fielding, [email protected], Akimasa Miyanaga, Bradley
S Moore.Center for Marine Biotechnology and Biomedicine, Scripps
Institution of Oceanography, La Jolla, CA 92037, United States
Production of the immunosuppressant lymphostin, a member of
the pyrroloquinoline alkaloid family of natural products, has been
observed in some species of the marine bacterium Salinispora.
Traditionally pyrroloquinolines have been isolated from sponges
or mushrooms; therefore, the genetic tractability of Salinispora,
along with genomic sequence data, has enabled exploration of the
biosynthetic origins of this class of compounds. Herein we describe
the elucidation of biosynthetic basis of lymphostin production in
Salinispora spp., which involves a uniquely organized polyketide
synthase-nonribosomal peptide synthetase hybrid. In addition,
gene targeting experiments led to the discovery of the novel
analogue lymphostinol.
BIOT 319
BIOT 317
Metabolic engineering of Taxus suspension cultures for
enhanced production of paclitaxel
Effect of TiO2 nanoparticles on Pre-adipocyte cell
differentiation
Sarah A. Wilson1, [email protected], Patricia Keen4, Jennifer
Chienhsiu Lin1, [email protected], Kayla Applebaum1, Marcia
Simon2, Miriam Rafailovich1. (1) Department of Material Science &
Engineering, Stony Brook University, Stony Brook, NY 11794, United States
(2) Department of Oral Biology & Pathology, Stony Brook University, Stony
Brook, New York 11794, United States
Titanium dioxide (TiO2) particles are widely used in all types of
personal care products. However, the effect of these particles on
adipose formation is still under question. In order to understand
the effect of the nanoparticles, we culture the pre-adipocyte cell
with TiO2 nanoparticles in the adipogenetic medium. We found
the adipose generation was hindered by the nanoparticles. The
rate of differentiation from the pre-adipocyte to adipocyte is lower.
Normanly2, Elsbeth L. Walker3, Joyce Van Eck4, Susan C. Roberts1.
(1) Department of Chemical Engineering, University of Massachusetts
Amherst, Amherst, Massachusetts 01003, United States (2) Department of
Biochemistry and Molecular Biology, University of Massachusetts Amherst,
Amherst, Massachusetts 01003, United States (3) Department of Biology,
University of Massachusetts Amherst, Amherst, Massachusetts 01003,
United States (4) Boyce Thompson Institute, Ithaca, New York 14853,
United States
In 2006, the FDA approved a plant cell culture process for
production of the anticancer drug paclitaxel; however, suspension
cultures have low product yields that are highly variable across cell
lines and over time within a single cell line, leaving considerable
room for process improvements. Superior paclitaxel-accumulating
Taxus lines may be generated through stable over-expression of
rate-influencing pathway genes or redirection of carbon flux away
from competing metabolic pathways. We have established a reliable
Agrobacterium-mediated stable transformation protocol for Taxus
suspension cultures. The following parameters were found to be key
for the consistent recovery of stable transformants: modification of
antioxidants used during Agrobacterium infection, optimization
of hygromycin concentrations in the selection medium and
delayed transfer of infected cells to selection medium until there
are signs of new callus growth. Utilizing this technology, we are
implementing overexpression and silencing strategies to overcome
bottlenecks to paclitaxel production by promoting carbon flux
through the paclitaxel biosynthetic pathway.
BIOT 320
Cell surface display-releasing system for enzymes directed
evolution using insoluble substrates
Zuoming Zhang1, [email protected], Yanyan Chen1, Wenying
Gao1, Hong Zhang1, Percival Zhang2. (1) Key Lab for Molecular
Enzymology and Engineering of Ministry of Education, Jilin University,
Changchun, Jilin 130012, China (2) Biological Systems Engineering
Department, Virginia Tech, Blacksburg, Virginia 24061, United States
One of challenges in enzymes dierected evolution is to design a
method to screen or select mutants by using nature insoluble
substrates. Cell surface display system shows a potential use in
overcome this challenge. However, the main weak of this system is
the displayed enzymes are immobilized on the cell surface and will
not freely interact to insoluble substrates in ager plate. To solve this
problem, we constructed two new cell surface display-releasing
systems. Intein was inserted between carrier (ice-nucleation
protein, INP) and enzymes in the first one which enzymes may
released from carrier by Intein-based protein self- cleavages.
To prevent spontaneous cleavage of Intein and increase carrier
capacity of the first system, we constructed a second cell surface
display system, in which the Intein position in the first system was
substituted by 11 aa of N-terminus Intein (IN). The C-terminus
of Intein was expressed separately and then was used to trigger a
site-specific cleavage at the N-terminuse of IN. We tested the above
two cell surface display systems by using red fluorescent protein as
reporter and all system showed high cleaving efficiencies. Two cell
surface display-releasing systems containing endo-glucanases have
been constructed. The clear halo zones formation on ager plate
containing regenerated amorphous cellulose (RAC) are on-going
to check. We suggest these two systems have high potential use in
endo-glucanase directed evolution.
121
Tuesday Afternoon
BIOT 313
Wednesday
Morning
Wednesday Morning Sessions
BIOT 321
Quorum sensing as a mechanism for the stable population
division in batch cultures
David N Quan1,2, [email protected], William E Bentley1,2. (1)
Bioengineering, University of Maryland College Park, College Park,
MD 20742, United States (2) Institute for Bioscience and Biotechnology
Research, College Park, MD 20742, United States
Understanding the development and stability/variability of
bacterial ecosystems (such as those found in biofilms, minimal
multispecies gut models, and endosymbiont-host relationships)
would be helpful in manipulating such bacterial communities
for therapeutic ends. Here we present the Lsr module (known to
import and process the quorum sensing molecule Autoinducer-2)
as a mechanism for generating a stable intraspecies population
division. A hybrid between the Lac operon and other quorum
signaling systems, the Lsr module does not regulate the production
of its own inducer, although the bacteria do synthesize and export
it. Building off of existing Lac system mathematical models, we
construct a model that describes Lsr system behavior. The model
has only a single steady state when modeling a unified population,
but multiple steady states when populations with different noise
terms compete against each other. This modeling may explain
apparently stable bimodal Lsr activity in E. coli batch growth.
A
B
C
8:30 a.m.
Downstream Processes: Bioseparation Fundamentals –
Modeling, Experiment, and Analytical Methods E. von Lieres, M. Ottens Papers 322-329
8:30 a.m Room# 16B
Upstream Processes: Protein Engineering – Methods and Applications
A.Link, A. Rakestraw Papers 330-336
8:30 a.m. Room# 17A
Upstream Processes: Microbial Process Development
J. Otero, K. Tyo Papers 337-344
8:30 a.m.
D
E
122
Room# 16A
Room# 25A Advances in Biotechnology Product Development: Manufacturing
Technical Support and Life Cycle Management of Biotechnology Products
K. Barnthouse, D. Adams Papers 345-352
Biophysical & Biomolecular Processes: Protein-X Interactions –
Aggregation in Disease and Therapeutics
I.Kwon, R. Latypov Papers 353-359
11:30 a.m. Room# 16A
8:30 a.m. Room# 25B
Elmer Gaden Award
Gregory J. Cost
123
Bioseparation Fundamentals – Modeling,
Experiment, and Analytical Methods 8:30 a.m.
Room# 16A
E. von Lieres, M. Ottens Papers 322-329
BIOT 322- 8:30 a.m.
Protein partitioning in aqueous two phase systems: A
molecular dynamics approach
Florian Dismer, [email protected], Stefan Oelmeier, Juergen
Hubbuch.Biomolecular Separation Engineering, Karlsruhe Institute of
Technology, Karlsruhe, Germany
Aqueous two-phase systems have proven to be a valuable alternative
for chromatographic separation steps in downstream processing
regarding selectivity and throughput. In this work molecular
dynamics simulations (MD) were used to get insights into system
properties leading to protein partitioning in PEG/phosphate
systems. MD simulations of single PEG molecules showed
increasing hydrophobicity with increasing molecular weight
correlating well with experimentally determined Et(30) values.
MD simulations for PEG/phosphate systems for different PEG
molecular weights (300 to 1500 Da) to determine phase-system
properties responsible for lysozyme distribution by linking MD
data to model parameters for the Abraham equation (polarizability
π*, solvent acidity α and basicity β) that was successfully applied
by Madeira and Zaslavsky for predicting partitioning coefficients.
A high-throughout screening was established using a robotic
system to determine α,β and π* using solvatochromic dyes. Data
was compared to simulation results and successfully used to build
a predictive model for lysozyme distribution.
BIOT 323 – 8:50 a.m.
Use of molecular simulations to understand and predict
multimodal ligand-protein interactions
Siddharth Parimal, [email protected], Melissa A Holstein, James A
Woo, Shekhar Garde, Steven M Cramer.Department of Chemical and
Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180,
United States
Molecular dynamics (MD) simulations have been employed to
establish a fundamental understanding of how multiple interactions
work together to create binding selectivity in chromatographic
124
systems. MD simulations were performed with increasingly
complex ligands to identify the key contributors to synergistic
interactions in multimodal (MM) ligand-protein systems. The
results were used to develop a new technique for mapping synergy
between electrostatic and hydrophobic regions on a protein surface
and for understanding avidity effects on resins to make predictions
of protein retention behavior in MM systems. Simulations were
also performed to quantify the effect of mobile phase modifiers
in modulating the different types of interactions in MM systems.
The knowledge base created using these simulations can be used
to select appropriate combinations of MM ligands and modifiers
to achieve unique selectivities for challenging protein separations.
BIOT 324 – 9:10 a.m.
Fast analytical tools applicable for protein purification process
development and monitoring
Sigrid Hansen1, [email protected], Stefan Oelmeier1, Patrick
Diederich1, Erik Skibsted2, Arne Staby2, Juergen Hubbuch1. (1)
Biomolecular Separation Engineering, Karlsruhe Institute of Technology,
Karlsruhe, Germany (2) Novo Nordisk A/S, Bagsværd, Denmark
Enhancement of process and product understanding is required
for implementing PAT and QbD, thus extensive, experimental
process characterization in high-throughput is required shifting
the bottleneck towards analytics.
Non-invasive, label-free selective protein quantification:
PLS regression was used to selectively correlate protein
concentrations to individual absorption spectra. On this basis a fast
and precise analytical assay was established for multi-component
mixtures.
Mab aggregate quantification in 90 s:
Combining interlaced injection with parallelization of two SEC
columns, lag times before and after the peaks of interest could
be eliminated. Aggregate levels and reproducibility were equal
to single injections and assay time could be pushed below two
minutes.
Evaluation of overlapping elution profiles:
To increase throughput in analytical chromatography, faster flow
rates, shorter columns or steeper gradients are necessary, however,
often at the expense of resolution. Here, PLS regression was used
to evaluate low resolution chromatograms. A 15-fold reduction of
analysis time was achieved.
BIOT 325 – 9:30 a.m.
Methodology for model based robustness analysis and design
of preparative protein chromatography
Bernt Nilsson1, [email protected], Karin Westerberg1,
Marcus Degerman1, Niklas Borg1, Arne Staby1,2. (1) Department of
Chemical Engineering, Lund University, Lund, Sweden (2) Novo Nordisk
A/S, Gentofte, Denmark
Model-based methodology for design and analysis of preparative
chromatography is becoming an interesting tool in pharmaceutical
industry, providing both a deep insight into the process behavior
and an efficient process design tool for quantitative predictions.
A methodology for quality by design is presented, which includes
identification of critical quality attributes and critical process
parameters and definition of design space for robust operation
and control, all based on mechanistic models and risk-based
assessment.
The model based approach allows complementing the
methodology with analysis of process performance attributes and
to perform process optimization with robustness constraints.
The methodology uses a calibrated mathematical model which
predicts column behavior. The mathematical chromatography
model has to contain descriptions of all variations of importance,
like changes in feed and mobile phase composition, changes in
operation and equipment parameters. The methodology also
includes methods to analyze model accuracy and its implications
on the robustness of the process.
BIOT 326 – 10:10 a.m.
Effect of titer on the development of an affinity
chromatography step
Andre C. Dumetz, [email protected], Adrian M. Gospodarek,
Jessica L. Lewis, Antonio R. Ubiera, David N. Paolella, Kent E. Goklen.
Downstream Process Development, GlaxoSmithKline, King of Prussia, PA
19406, United States
Affinity chromatography for the capture of a biopharmaceutical,
when available, is generally preferred because of its selectivity.
Column loading optimization is thereafter an important issue
because of resin cost. In this work, the Dynamic Binding Capacity
(DBC) as a function of protein concentration is reported for
commercially available Protein A resins and three proteins of
different sizes. A 10-40% increase in the measured DBC is observed
when the protein concentration in the feed increases from 1g/L to
5g/L. This behavior was investigated by measuring static binding
capacity and diffusivity. The shape of the binding isotherm can be
fitted using the colloidal isotherm and used to explain the increase
in DBC observed experimentally. The accessible surface area was
determined using inverse size exclusion chromatography to show
that commercially available Protein A resins are optimized for
IgGs, but not for smaller protein molecules.
BIOT 327 – 10:30 a.m.
Model-based chromatographic resin selection
Beckley K. Nfor, Diego S. Zuluaga, Peter J.T. Verheijen, Luuk A.M. van
der Wielen, Peter D.E.M. Verhaert, Marcel Ottens, m.ottens@tudelft.
nl.Biotechnology, Delft University of Technology, Delft, The Netherlands
In this work, a model-based rational strategy for the selection of
chromatographic resins was proposed and illustrated by evaluating
three mixed mode adsorbents for the separation of a ternary mixture
of BSA, ovalbumin and amyloglucosidase. The main question
addressed was selecting the most optimal chromatographic
resin from a few promising alternatives. The methodology starts
with chromatographic modeling, parameters acquisition and
model validation, followed by model-based optimization of the
chromatographic separation for the resins of interest. Finally, the
resins are rationally evaluated based on their optimized operating
conditions and performance metrics such as product purity, yield,
concentration, throughput, productivity and cost. The proposed
model-based approach could be a suitable alternative to column
scouting during process development, the main strengths being the
fact that resins are evaluated under their ideal working conditions,
enabling a fair comparison. This work also demonstrates the
application of column modeling and optimization to mixed mode
chromatography.
BIOT 328 – 10:50 a.m.
Chromatography modeling at bead level for bioprocess design
and understanding
Spyridon Gerontas, [email protected], Michael S. Shapiro, Daniel
G. Bracewell.Department of Biochemical Engineering, University College
London, London, United Kingdom
Chromatographic modeling can be used as a predictive tool for
the investigation of different design and operating conditions
with minimal experimental effort. Current modeling approaches
assume uniform rate parameters throughout the column. A new
modeling approach to describe chromatographic processes in a
greater detail is therefore required. Software and hardware advances
now allow us to consider what can be learnt from modeling at
bead level. In this study a model has been developed to simulate
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Wednesday Morning
Downstream Processes:
BIOT 329 – 11:10 a.m.
Model-based scale-up with the zonal rate model of membrane
chromatography
Pranay Ghosh1, Min Lin2, Jens Vogel2, Eric von Lieres1, e.von.
[email protected]. (1) Institute of Bio- and Geosciences 1, Research
Center Juelich, Juelich, NRW 52425, Germany (2) Global Biological
Development, Bayer Healthcare, Berkeley, CA 94701, United States
Mass transfer in membrane chromatography is predominantly
through convection, which facilitates higher flow rates and higher
throughput as compared to packed bed chromatography. However
early breakthrough and unfavorable peak tailing can be caused by
inhomogeneous bulk flow when membrane sheets are stacked in
modules with extreme length to width ratios. The same applies for
spiral wound membranes in radial flow modules.
Fluid dynamics external to the membrane must be considered
when modeling transport and sorption within the membrane.
Membrane chromatography is usually modeled in one spatial
dimension, assuming homogeneity over membrane cross sections,
which is practically hard to achieve in membrane capsules.
The previously published Zonal Rate Model (ZRM) allows to
quantitatively analyze the impact of inhomogeneous membrane
loadings on measured chromatograms by virtually partitioning
hold-up volumes and the membrane stack. The binding parameters
can be transferred across geometries for model-based scale up and
process design.
Upstream Processes:
Protein Engineering – Methods and
Applications
8:30 a.m Room# 16B
A.Link, A. Rakestraw Papers 330-336
BIOT 330 – 8:30 a.m.
Computational model of VDJ recombination for antibody
design
have developed an integrated approach that combines systems
biology-driven design to reveal optimal inhibitor characteristics
and to identify independent engineering modules with rapid
antibody optimization platform. We implemented focused yeast
display selection protocols to optimize antibody modules for high
affinity and robust pharmaceutical properties in a single campaign.
We showed that these modules retain their optimal properties both
as soluble proteins and as components of multi-specific antibodylike molecules. Here, we present an application of this approach
to optimization of a bispecific molecule targeting two receptor
tyrosine kinases.
BIOT 332 – 9:10 a.m.
Robert J Pantazes, [email protected], Costas D Maranas.
Rapid isolation and production of antibody-like reagents using
a cloning-free yeast surface display approach
United States
James A. Van Deventer1, [email protected], Gabriela Pregernig3,
Chemical Engineering, Penn State University, University Park, PA 16802,
The ability of antibodies to activate the immune system by binding
antigens has made them an important and rapidly growing class
of medications in the fight against cancer and other diseases.
Antibodies are assembled in the human immune system by
randomly recombining variable, diversity, and joining genes in a
process known as VDJ recombination. In this work, we introduce
a computational model of VDJ recombination that can predict an
antibody’s structure from its primary sequence. We created this
model by predicting structures for each known variable, diversity,
and joining gene in the human genome, encompassing ~108 possible
antibodies. The model is used, in conjunction with OptCDR, to
computationally suggest how to fully humanize existing antibodies
as well to de novo design fully-human antibodies against any
specified epitope.
BIOT 331 – 8:50 a.m.
Rapid optimization of antibody modules to enable
construction of multi-specific antibody therapeutics.
Sachdev S. Sidhu2, Karl D. Wittrup1,3. (1) Department of Chemical
Engineering, Massachusetts Institute of Technology, Cambridge, MA
02139, United States (2) Department of Medical Research, University of
Toronto, Toronto, Ontario M5S 3E1, Canada (3) Department of Biological
Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139,
United States
Display technologies form an integral part of protein engineering,
but their implementation remains limited by the rigorous
molecular biology, protein production, and protein purification
steps involved in the development of engineered proteins. Here we
present an approach that streamlines the isolation of antibody-like
reagents by integrating display and secretion capabilities within
a yeast-based library of single chain variable fragment-constant
region (scFv-Fc) proteins. During screening, the scFv-Fc proteins
are displayed on the surface of yeast via fusion to the Aga2p protein,
while manipulation of medium conditions enables secretion of
soluble scFv-Fc proteins. Initial results indicate modest levels of
scFv-Fc display on the yeast surface and secretion yields of two to
five milligrams per liter. We expect that our approach will enable
the routine isolation of affinity reagents by many researchers
and be useful for biological assays such as western blotting, flow
cytometry, and ELISA assays.
Yang Jiao, [email protected], Lihui xu, Rachel Rennard,
Neeraj Kohli, Maya Razlog, Bryan Johnson, Jian Tang, Jason Baum, Ulrik
Nielsen, Birgit Schoeberl, Jonathan Fitzgerald, Alexey Lugovskoy.antibody
technology, merrimack pharmaceuiticals, cambridge, MA 02139, United
States
Multi-specific antibody-like molecules capable of blocking several
receptor signaling pathways and recruiting adaptive immune
functions represent a promising new class of anti-cancer agents. Yet,
their construction has faced significant challenges due to the large
number of interlinked optimization parameters, such as multiple
affinities, avidities and CMC properties. To solve this problem we
126
**Extended Intermission 9:30 – 10:10 a.m.**
BIOT 333 – 10:10 a.m. **Talk starts 20 minutes later than
what the ACS Program states**
SECANT® yeast display platform for discovery of human
antibodies, engineering of complex scaffolds, and novel
selections of protein therapeutics
Andy Rakestraw, [email protected] LLC,
Cambridge, MA 02139, United States
The Secretion and Capture Technology (SECANT®) platform
utilizes a non-covalent cell surface attachment to display a protein of
interest on the surface of yeast for protein engineering applications.
With this method, diversified libraries of proteins including
complex scaffolds (full-length IgG, Ig derivatives, multispecific
antibodies, binding-scaffolds, etc.) can be constructed and
screened for binding and non-binding attributes such as improved
expression, stability, solubility, reduced aggregation, etc via highthroughput flow cytometry or magnetic-bead sorting.
We describe the novel aspects of the SECANT® platform in
conjunction with our fully-human, IgG1 Library for de novo
discovery of therapeutic antibodies. Additionally, we describe
how the platform has been used to construct and screen a tandem
scFv antibody library incorporating novel forms of diversity for
improved binding to a target. We also demonstrate how the unique
nature of the SECANT® platform facilitates the purification and
characterization of mutant clones directly from the selection
output.
BIOT 334 – 10:30 a.m. **Talk starts 20 minutes later than
what the ACS Program states**
Structure-based design of conformation-specific antibodies
against misfolded proteins
Joseph M Perchiacca, Ali Reza A Ladiwala, Moumita Bhattacharya,
Peter M Tessier, [email protected] of Chemical & Biological
Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, United
States
Conformation-specific antibodies that recognize aggregated
proteins associated with several conformational disorders (e.g.,
prion diseases) are invaluable for diagnostic and therapeutic
applications. However, no systematic strategy exists for generating
conformation-specific antibodies that target linear sequence
epitopes within misfolded proteins. We have developed a novel
approach for designing conformation- and sequence-specific
antibodies against misfolded proteins that is inspired by the
homotypic interactions governing protein aggregation. We
demonstrate that grafting small hydrophobic peptides from
several amyloidogenic proteins (e.g., Aβ42 peptide associated with
127
Wednesday Morning
at bead level protein loading in 1.5 µl microfluidic columns. It
takes into account the heterogeneity in bead sizes and the spatial
variations of the characteristics of a packed bed, such as porosity
and dispersion, thus offering a detailed description of the flow field
and mass transfer phenomena. Simulations were shown to be in
good agreement with published experimental data demonstrating
a comprehensive process understanding which could be proven
useful in predicting the large scale.
BIOT 335 – 10:50 a.m. **Talk starts 20 minutes later than
what the ACS Program states**
Optimization algorithms for the design of immunotolerant
biotherapies
proteins binders, and show the application of the method for the
de novo design of two separate proteins that bind to a neutralizing
epitope of the 1918 H1N1 pandemic Influenza virus. Structures
of both designs (after affinity maturation) in complex with their
target reveal that the actual binding interface is nearly identical
to that in the computational design model. Improvements to
the design process were sought. To that end, I will highlight a
community-wide assessment of our designed proteins that identify
several deficiencies in the computational design process. On the
experimental side, we used next-generation sequencing coupled
to yeast display selection to determine the effects of every single
amino acid substitution on binding of our designs to multiple HA
subtypes. Screening of a library containing beneficial substitutions
identifies an improved design that neutralizes Influenza viruses.
The explosive growth of biotherapeutic agents is revolutionizing
treatment of numerous diseases, but innovations in biotherapies
have also created new challenges for drug design and development.
One distinguishing risk factor of therapeutic proteins is the
prospect of eliciting an immune response in humans. To meet
this challenge, we have developed optimization algorithms that
minimize a protein’s T cell epitope content while simultaneously
ensuring that the engineered variant maintains a high level of
stability and activity. Our algorithms assess immunogenicity using
T cell epitope predictors that score peptide binding potential
to class II MHC molecules. The structural and functional
consequences of deimmunizing mutations are evaluated with
statistical sequence potentials and molecular mechanics force
fields. The development and implementation of these algorithms
will be highlighted through comparative analysis with previously
published deimmunization efforts as well as our own experimental
validation using beta-lactamase, a model therapeutic candidate
with utility in ADEPT cancer therapies.
BIOT 338 – 8:50 a.m.
Engineering terpenoid natural product biosynthesis in
Escherichia coli
Ajikumar Parayil1,2, [email protected], Chin Giaw Lim1, Marjan De
Mey1, Gregory Stephanopoulos1. (1) Chemical Engineering, Massachusetts
Institute of Technology, Cambridge, MA 02139, United States (2) Manus
Biosynthesis Inc., Cambridge, MA 02139, United States
Andrew S. Parker1, [email protected], Daniel C. Osipovitch1,
Joseph Desrosiers2, Lu He1, Annie De Groot2, Karl E. Griswold1, Chris
Bailey-Kellogg1. (1) Dartmouth College, United States (2) University of
Rhode Island, United States
rapidly enriched, with nearly 40% and 300% improved growth
rates over the WT in minimal medium supplemented with 2.5%
or 5% v/v ethanol, respectively. The roles of the over-expressed
genes in the resistance phenotypes, including the kinetics of
glucose consumption and ethanol production, growth in fed-batch
cultures, cell size homeostasis, and viability will be presented.
Upstream Processes:
Microbial Process Development
8:30 a.m. Room# 17A
J. Otero, K. Tyo Papers 337-344
BIOT 337 – 8:30 a.m.
Discovery of gene over-expression targets for improved ethanol
resistance in Saccharomyces cerevisiae
Pedro V Peña1, [email protected], Steven Glasker2, John
Hundertmark2, Jared Cohen2, Friedrich Srienc1,2. (1) Biotechnology
Institute, University of Minnesota, St. Paul, MN 55108-6106, United States
(2) Department of Chemical Engineering and Materials Science, University
Most biologically-active natural products are heavily substituted
molecules with multiple chiral centers. Unfortunately, the structural
complexity of natural products precludes the development of
economical synthetic routes to these molecules and procuring
marketable quantities of these substances from their natural
sources is equally challenging. These supply bottlenecks have
restricted their wide applications. Our recent research in metabolic
engineering of terpenoid biosynthesis in E. coli offers a new
paradigm for commercially exploiting terpenoid natural products
for therapeutics and other applications. The present study reports
the metabolic engineering of diterpenoid taxadiene and kaurene in
E. coli. Our multivariate modular metabolic engineering approach
enabled the systematic assessment and elimination of regulatory
and pathway bottlenecks by re-defining the metabolic network as
two modules for taxadiene and kaurene. We have extended our
approach up to three modules for the engineering of hydroxylated
diterpenoids and established that achieving the fine balance in the
pathway expression is key for the high level production of these
molecules. The present talk will be focused on the development
of our new metabolic engineering approach, MVME and its
application to the production of complex diterpenoids.
of Minnesota, United States
BIOT 336 – 11:10 a.m. **Talk starts 20 minutes later than
what the ACS Program states**
Leave no amino acid unturned: Computational de novo design
and fitness landscape exploitation leads to potential influenza
therapeutics
Tim A Whitehead, [email protected] Engineering and
Materials Science, Michigan State University, East Lansing, MI 48824,
United States
I will present a general method for the computational design of
128
Ethanol induces one of the most potent stresses faced by
Saccharomyces cerevisiae during industrial processes to produce
this biofuel. Thus, engineering of resistance to ethanol is essential
to improve yields and process efficiency. Here, the Cytostat
cell culture technique, developed in our lab, is used to screen
for ethanol resistance phenotypes in cells transformed with a
genome-wide, gene over-expression library. The Cytostat selects
for inhibitor specific resistance because the continuous culture is
maintained at very low cell densities, and thus the selective pressure
remains constant. Further, the Cytostat selects for the fittest, most
resistant clone. In this way, ethanol resistant transformants were
BIOT 339 – 9:10 a.m.
Process understanding approach for a late-stage recombinant
protein vaccine produced in Saccharomyces cerevisiae
recombinant protein vaccine produced in Saccharomyces cerevisiae.
The manufacturing process consists of glucose batch and glucoselimited exponential fed-batch growth phases and a galactoseinduced protein production phase. Physiological characterization
was combined with transcriptomic and extracellular metabolomic
analyses to elucidate the observed lack of galactose metabolism
of the strain. When grown on various carbon sources, including
glucose, galactose, and ethanol, the strain only grew well on glucose.
During continuous cultivation, the strain washed out at a dilution
rate of 0.2 h-1, which is significantly lower than the batch glucose
maximum specific growth rate (0.29 h-1). Transcriptome analysis
revealed no dysfunction in transcription of any of the major
galactose metabolic pathway genes. This work demonstrated the
value of a process understanding approach, which can be leveraged
for improvements to existing manufacturing-scale processes or de
novo process development programs.
BIOT 340 -9:30 a.m.
Efficient process for propionic acid production from renewable
feedstock using engineered propionibacteria
Zhongqiang Wang, [email protected], Shang-Tian Yang.
Chemical & Biomolecular Engineering, The Ohio State University,
Columbus, OH 43210, United States
Propionibacterium freudenreichii subsp. shermanii, commonly
used in industrial cheese and vitamin B12 production, was
engineered to produce propionic acid with high yield and
productivity. In propionic acid fermentation, pyruvate as a central
metabolite is converted to propionic acid through the WoodWerkman cycle or to acetic acid through the pta-ack pathway.
Methylmalonyl-CoA transcarboxylase plays a crucial role in the
Wood-Werkman cycle: carboxylation of pyruvate and formation
of the precursor of propionic acid. In this study, this enzyme
was overexpressed in P. shermanii to divert more pyruvate to the
NADH-consuming propionic acid pathway in fermentation using
glycerol and glucose as the co-substrates. Compared with the
wild type, propionic acid yield and productivity were significantly
increased in the mutant. The propionate/acetate ratio was also
elevated due to the shifted carbon flux. The characterization of the
engineered mutant and its fermentation kinetics compared to the
wild type will be presented in this paper.
Amanda Schallop1, [email protected], Brian P
Doyle1, [email protected], Nedim Altaras1, Brian E Mickus2,
José M Otero1. (1) Vaccine Process Development, Merck & Co., Inc., West
Point, PA 19486, United States (2) Molecular Profiling and Research
Informatics, Merck & Co., Inc., West Point, PA 19486, United States
A process understanding approach was applied to a late-stage
129
Wednesday Morning
Alzheimer’s disease) into the complementarity determining regions
of small antibodies generates antibody variants that selectively
recognize aggregated proteins containing the cognate peptide
motifs (Perchiacca et al., PNAS, 2011). We refer to these antibodies
as gammabodies for Grafted AMyloid-Motif AntiBODIES. We
will discuss how gammabodies can be designed against diverse
misfolded proteins, as well as how they can be used to probe the
structures of misfolded proteins in a site-specific manner.
BIOT 343 – 10:50 a.m.
Expression of recombinant proteins by a novel
thermoinducible two-compartment continuous production
system
High-titer n-butanol synthesis in Clostridium tyrobutyricum
with external driving forces
Octavio T. Ramirez, [email protected], Oriana L. Niño,
of Chemical and Biomolecular Engineering, The Ohio State University,
Nahandi A. Tepez.Instituto de Biotecnologia, Universidad Nacional,
Mexico, Mexico
Yinming Du, [email protected], Shang-Tian Yang.Department
Columbus, Ohio 43210, United States
Thermoinduction is among the most commonly used induction
strategies for production of recombinant proteins as it yields high
expression levels, is simple and avoids the introduction of toxic
or costly chemical inducers. Yet, traditional thermoinduction
results in growth cessation due to the high metabolic load caused
by heterologous protein production and the stress caused by
the heat shock response. In this work we will show that a twocompartment chemostat system can be used for continuously
producing a recombinant pr-proinsulin by Escherichia coli. In such
a system, cells are continuously recirculated between high and low
temperatures maintained in each compartment, while fresh media
is continuously fed whereas metabolized broth is continuously
withdrawn. Such a system allowed the evaluation, for the first time,
of the effect of growth rate on thermoinduction of a recombinant
protein. Kinetic and stoichiometric data, within a broad range of
dilution rates, will be presented.
The biosynthesis of n-butanol through aldehyde/alcohol
dehydrogenase (adhE2) is limited by NADH availability and the
butanol titer. To alleviate these limitations and increase n-butanol
production in C. tyrobutyricum-adhE2 mutants, increased
NADH driving force via the addition of an electron carrier such
as methyl viologen in the culture medium, which significantly
improved NADH availability and directed higher carbon flux into
the n-butanol synthesis pathway, and continuous gas stripping to
remove butanol in situ, which reduced butanol inhibition, were
applied to achieve high-titer butanol production from glucose
in both batch and fed-batch fermentations. Compared to the
control without these external driving forces, butanol production
increased more than five-fold to >50 g/L (vs. <10 g/L) and both
acetic and butyric acids production were reduced to less than 2 and
5 g/L, respectively. Butanol yield was also significantly increased
from 0.10 g/g to 0.30 g/g glucose.
BIOT 342 – 10:30 a.m.
BIOT 344 – 11:10 a.m.
Process development for a new R1-based plasmid DNA vaccine
vector
Optimization of diploid Pichia pastoris strains for the
expression of full-length monoclonal antibodies
Diana M Bower, [email protected], Kristala LJ Prather.Department
Leon F. Garcia-Martinez, [email protected]
of Chemical Engineering, Massachusetts Institute of Technology,
Cambridge, MA 02139, United States
Recent renewed interest in plasmid DNA (pDNA) as a therapeutic
has increased the demand for robust, high-yield manufacturing
platforms. To help meet this demand, we have developed a new
DNA vaccine vector, pDMB02-GFP, containing a runaway R1
origin of replication. This origin typically gives a temperatureinduced increase in plasmid copy number, but we have observed
that pDMB02-GFP often produces higher yields without a
temperature shift. In shake flasks at 30°C, our vector achieved
yields of approximately 15 mg pDNA/g dry cell weight of highlysupercoiled product. Ongoing work is focused on designing
a bioreactor-scale process for production of pDMB02-GFP.
We have identified several scale-up challenges, including the
need for growth rate control. Additional work seeks to better
understand the mechanism underlying replication of R1-based
vectors in industrially-relevant culture conditions and to use this
understanding to guide process design.
130
Biopharmaceuticals, Bothell, WA 98011, United States
We developed a rapid, robust technology platform for production
of full-length humanized monoclonal antibodies in Pichia pastoris.
This expression system captures all the attributes provided by this
host (low cost, strain stability, etc.) while eliminating product
development risk associated with untested expression systems. The
platform has been successfully run under GMP at 2000L scale with
excellent reproducibility. Our first molecule, ADL518 has already
been successfully used in human clinical trials. In this presentation
a novel approach for the generation of diploid Pichia pastoris
strains with increased yield and purity will be discussed.
Advances in Biotechnology
Product Development:
Manufacturing Technical Support and Life
Cycle Management of Biotechnology Products
8:30 a.m.
Room# 25A K. Barnthouse, D. Adams Papers 345-352
control software. It is based on the combinations of various types
of feedback control.
BIOT 347 – 9:10 a.m.
Modernization of the downstream purification process for a
commercially-approved monoclonal antibody product
Frank Maslanka, [email protected] pharmaceutical company
of Johnson & Johnson, Pharmaceutical Development and Manufacturing
Sciences, Janssen Pharmaceuticals Inc., United States
BIOT 345 – 8:30 a.m.
Overcoming technical challenges while updating a legacy
commercial production process
Rick St. John, [email protected], Jane Gunson, Wendy Lau,
Mike Laird.Purification Development, Genentech, United StatesLate Stage
Cell Culture, Genentech, United StatesGenentech, United States
A legacy Genentech process was updated to increase manufacturing
flexibility, ensure product supply, and improve operator safety. Cell
culture productivity was increased 2-fold and the purification
process was revamped to improve plant fit, remove a prominent
product variant and increase throughput. Only the cell bank and
drug product formulation / configurations were maintained.
Several example technical challenges during development and
implementation will be discussed. The final process utilizes
an entirely different purification process flow, but analytical
comparability was successfully achieved.
BIOT 346 – 8:50 a.m.
In-line buffer conditioning for monoclonal antibody
manufacturing
Enrique Carredano, [email protected], Tomas M Karlsson,
Roger Nordberg, Gustav Rodrigo, Elenor Strandberg, Henrik Sandegren.LSBio Technologies, GE Healthcare, Uppsala, Sweden
Buffer preparation and handling is a major challenge in industrial
monoclonal antibody production. There is a need to decrease
costs in labour and space, this need can be met by formulating
the buffers in-line using concentrated stock solutions and water
for injection (WFI). In-line Conditioning (IC) is GE Healthcare’s
newly introduced concept for buffer formulation combined with
a purification step such as chromatography or filtration in large
scale, based on the ÄKTA™ process platform with UNICORN™
While the drivers for this process modernization included improved
robustness, reduction in cycle time and reduction in cost of goods,
modernizing a commercially-approved process for a legacy product
carries the potential for risk, challenges and complexity throughout
development, implementation and validation. The modernization
of the downstream purification process for a monoclonal antibody
product involved implementing dead-end virus filtration in place
of tangential flow virus filtration, modifying a process stage and
relocating the virus filtration step in the process. The successful
development, scale-up, implementation and validation of these
modifications to the downstream purification process will be
discussed.
BIOT 348 – 9:30 a.m.
Critical reagent supply as an essential element in product
management: A case study
Michele M Myers1, [email protected], Cristy Dougherty1,
Paul Boyd2, Saroj Ramdas1, Robert Ryland1, John Erickson1. (1) Global
Manufacturing & Supply, GlaxoSmithKline, King of Prussia, PA 19406,
United States (2) Global Manufacturing & Supply, GlaxoSmithKline,
Stevenage, United Kingdom
Critical reagent supply is often overlooked as part of product
lifecycle management until something goes wrong. The process for
producing a biological reagent required for routine product release
testing had worked for many years. However, because the reagent
production process was never completely characterized, response
to out of specification reagent required significant resources and
time. Failures in reagent production threatened supply of drug to
patients so short, medium, and long term strategies had to be used
in parallel to manage reagent supply. First, the consumption of
available reagent was prioritized to testing related to product release
only. Next, an alternative assay method was developed and qualified
using the out of specification reagent. These interim strategies
bought time while the root cause investigation was conducted and
131
Wednesday Morning
BIOT 341 – 10:10 a.m.
BIOT 349 – 10:10 a.m.
Providing a recipe framework to aid in lifecycle support of
large molecules
Adam M Fermier1, [email protected], Steve J Mehrman1, Danielle
Higgins2, Nathan Skacel1, John Cunningham1, Maaike Poppema3, Shaun
McWeeney1, Walter Cedeno4, John Stong4, Andrew Skalkin4, Terry
Murphy1. (1) Pharmaceutical Development and Manufacturing Sciences,
Janssen Pharmaceuticals Inc., a pharmaceutical company of Johnson &
Johnson, Spring House, Pa 19477, United States (2) Janssen Supply Chain,
Janssen Pharmaceuticals Inc., a pharmaceutical company of Johnson &
Johnson, Malvern, PA 19355, United States (3) Janssen Supply Chain,
Janssen Pharmaceuticals Inc., a pharmaceutical company of Johnson
& Johnson, Leiden, Einsteinweg 101 071 5242444, Netherlands Antilles
(4) Informatics Center Of Excellence, Janssen Pharmaceuticals Inc., a
pharmaceutical company of Johnson & Johnson, Spring House, PA 19477,
United States
Commercial biopharmaceutical products provide unique challenges
in lifecycle support due to the complexity of the manufacturing
process; in particular the basics of material traceability through the
plant can be quite intricate. To help overcome this complexity, a
scalable recipe based data warehousing strategy is being adopted to
help on such activities. The strategy can help on new investigations
and has proven valuable in aggregation of additional process and
product characterization data that can span multiple systems. In
the presentation, we will present the importance of the recipe
structure our plans to use this for workflow execution in MES
and LES and collect and aggregate data from typical Enterprise
Resource Planning systems (ERP), lab information systems (LIMS),
Process Data Historian systems and custom databases. We will also
demonstrate how bringing this information into a consolidated
location can facilitate the transfer from implicit knowledge to tacit
knowledge around processes.
BIOT 350 – 10:30 a.m.
Implementation of redundant sterile filtration process for bulk
and final fill operations
Ranjeet S Patil, [email protected], Michael J Felo, Chase
redundant filtration for final product sterilization. Regulatory
requirements for redundant filtration have become more stringent
in the past decade and can vary significantly by geographic region.
This creates a moving target for regulatory compliance for new and
existing products being sold or manufactured in new markets. Long
processing times, high product value, and the risk of reprocessing
in case of filter integrity test failure during final fill operations are
also practical drivers for the adoption of redundant filtration.
The operational complexity of using sterile filters in series can
make the implementation of this process difficult. For this study,
the strategy and execution of post-sterilization integrity testing of
filters, before and after use, are examined for regulatory compliance
and minimization of risk to the process. Other key implementation
aspects such as filtration train sizing and flushing requirements for
extractables and leachables are reviewed. Regulatory compliance
requirements from different regions were reviewed, and their
implications for system design and process sequence are evaluated.
Finally, the ability and potential benefits of single-use assemblies
to meet the operational and regulatory demands of redundant
filtration are considered.
BIOT 351 – 10:50 a.m.
Using relational modeling as a tool for data management in
bioprocesses: A case study
Bill Henry, Bruce Vickroy, Chittoor Narahari, chittoor.r.narahari@
gsk.com.Global Manufacturing and Supply, GlaxoSmithKline, 709
Swedeland Road, King of Prussia, PA 19406, United States
Even though online data (such as pH trends in a bioreactor or
UV traces in chromatography columns) are easily accessible,
offline data (such as analytical results) are still needed to tell the
complete story of a batch. A method for organizing and managing
online and offline data associated with a biopharmaceutical drug
substance batch will be presented. This method is based on a
relational model of the workflow involved in a typical monoclonal
antibody process. We will discuss why relational databases are a
useful tool for this purpose and provide applications of this tool
for data trending, periodic product reviews, investigation of
deviations, etc. Designing and implementing the relational model
requires investment up front, but it makes data and knowledge
management more effective than spreadsheets or paper.
BIOT 352 – 11:10 a.m.
Process transfer of an improved CHO cell culture process with
novel processing conditions to achieve comparable product
quality attributes
Christina T. Petraglia, [email protected], Angela Meier, Kara
Calhoun, Steven Meier.Late Stage Cell Culture - Pharma Technical
Development, Genentech, South San Francisco, California 94080, United
States
As part of Genentech’s life cycle management, a commercial
non-antibody CHO cell culture process was improved. This
case study will discuss the development and transfer to fullscale manufacturing throughout development, engineering
and qualification campaigns. The updated cell culture process
eliminates animal-derived hydrolysates, improves yield and
robustness, and simplifies complex operations. Although most
product quality attributes were comparable, early development
of the new process generated material with more mature glycans,
particularly increased sialic acid content. Based on knowledge of
glycosylation mechanisms, comparable sialic acid content was
achieved using low pH conditions in the cell culture process that
releases and activates sialidase, an endogenous CHO enzyme.
This process has been successfully demonstrated at multiple sites
and scales. Challenges and solutions to implementing the low pH
conditions at full-scale will be discussed. This new and improved
process resulted in a higher-titer process with comparable product
quality.
Biophysical & Biomolecular
Processes:
Protein-X Interactions – Aggregation in
Disease and Therapeutics
8:30 a.m. Room# 25B
I.Kwon, R. Latypov Papers 353-359
BIOT 353 – 8:30 a.m.
Duclos-Orsello.Biomanufacturing Sciences Network, EMD Millipore,
Billerica, Massachusetts 01821, United States
Protein condensation, liquid–liquid phase separation, and
human disease
Due to the potential risks of using filtration as a method of
sterilization, many regulatory agencies are requiring the use of
Aleksey Lomakin1, [email protected], Ying Wang1, George B
Benedek1,2. (1) Materials Processing Center, Massachusetts Institute
of Technology, Cambridge, Massachusetts 02139, United States (2)
132
Department of Physics, Massachusets Institute of Technology, Cambridge,
MA 02139, United States
Liquid-liquid phase separation is often not directly evident in
solutions of globular proteins, nevertheless it is a metastable
phenomenon underlying other forms of protein condensation,
such as aggregation, gelation and crystallization. Of all the protein
condensations, liquid-liquid phase separation is the least sensitive
to the detailed features of the inter-protein attraction and therefore
is most amenable to quantitative phenomenological analysis. On the
basis of our experimental and theoretical investigations of liquidliquid phase separation in solutions of gamma-crystallins and
immunoglobulins we will discuss: (i) the factors which determine
the location of the critical point and the shape of the coexistence
curve; (ii) use of the phase diagram as a tool for evaluating the
effect of solution conditions and the presence of other solutes
on the net interprotein attraction, and the consequent stability
of protein solutions; (iii) examples of human diseases (cataract,
cryoglobulemia) associated with liquid-liquid phase separation
and protein condensation.
BIOT 354 – 8:50 a.m.
Phase separation in solutions of monoclonal antibodies and
the effect of human serum albumin
Ying Wang1, Aleksey Lomakin1, Ramil F Latypov2, rlatypov@amgen.
com, George B Benedek1. (1) Materials Processing Center, Massachusetts
Institute of Technology, Cambridge, MA 02139, United States (2)
Department of Process and Product Development, Amgen Inc., Seattle, WA
98119, United States
We report the observation of liquid-liquid phase separation in
a solution of human monoclonal antibody, IgG2, and the effects
of human serum albumin, a major blood protein, on this phase
separation. We find a significant reduction of phase separation
temperature in the presence of albumin, and a preferential
partitioning of the albumin into the antibody-rich phase. We
provide a general thermodynamic analysis of the antibody-albumin
mixture phase diagram and relate its features to the magnitude of
the effective inter-protein interactions. Our analysis suggests that
additives (HSA in this report), which have moderate attraction
with antibody molecules, may be used to forestall undesirable
protein condensation in antibody solutions. Our findings are
relevant to understanding the stability of pharmaceutical solutions
of antibodies and the mechanisms of cryoglobulinemia.
133
Wednesday Morning
a long term solution implemented. This multipronged approach to
management of reagent supply was successful in avoiding an outof-stock situation for the drug product and provided numerous
lessons learned for product management teams.
Unfolding Fab domains via high temperature MD simulations:
Insights into aggregation liabilities
Patrick Buck, [email protected], Sandeep Kumar.
Biotherapeutics, Pfizer, Saint Louis, MO 63141, United States
Thioflavin T binding with mAb aggregates suggests the presence
of amyloid fibril forming sequence motifs called AggregationProne Regions (APRs). Furthermore, several APR motifs are often
predicted within mAb sequences. However, differentiating which
predicted APRs are playing an active role in aggregation requires
an understanding of the conformational changes that molecules
undergo in response to environmental stress. Analogous to
accelerated stability test, we conducted several high temperature
molecular dynamics simulations to monitor the structural changes
during the unfolding of Fab molecules. Predicted APRs were
localized to β-strands at the interfaces between structural domains
and in CDRs as well as adjacent framework regions. APRs motifs in
interfacial strands remain stable and solvent protected longer than
all other APRs. Thus, these sequence regions are effectively blocked
from promoting aggregation. Efforts to reduce mAb aggregation
should, therefore, focus on APRs that lie outside domain interfaces
e.g. CDRs and adjacent framework regions.
BIOT 356 – 9:30 a.m.
Highly aggregated antibody therapeutics can enhance the
innate and late-stage T-cell immune responses
Marisa Joubert1, [email protected], Martha Hokom2, Catherine
Eakin5, Lei Zhou3, Meghana Deshpande2, Matt Baker4, Terry Goletz2,
Bruce Kerwin5, Naren Chirmule2, Linda Narhi1, Vibha Jawa2. (1)
Department of Product and Process Development, Amgen, Thousand Oaks,
CA 91320, United States (2) Department of Clinical Immunology, Amgen,
United States (3) Department of Medical Sciences, Amgen, United States
(4) Antitope, United Kingdom (5) Department of Product and Process
Development, Amgen, Seattle, WA, United States
Aggregates of protein therapeutics have the potential to induce
an immunogenic response. Here, a set of aggregated therapeutic
antibodies (IgG1 and IgG2), known to have widely varying
properties, were shown to enhance the innate immune response
of a population of human peripheral blood mononuclear cells
(PBMC). This response appeared to depend on the aggregate type,
inherent immunogenicity of the molecule, and responsiveness
of the donor. This response also required high particle counts, at
least in this in vitro system, well above that detected in marketed
drug products. We propose a cytokine signature as a biomarker
of the PBMC response to aggregates. The cytokines identified
include: IL-1β, IL-6, IL-10, MCP-1, MIP-1α, MIP-1β, MMP-2 and
134
TNF-α. Aggregates made by stirring induced the highest response
compared to aggregates made by other stress methods. Particle size
in the 2 -10 μm range and the maintenance of at least some partially
folded structure were associated with an increased innate response.
The mechanism of aggregate activation at the innate phase was
found to occur through specific cell surface receptors (the toll-like
receptors TLR-2 and TLR-4, FcγRs, and the complement system).
The innate signal was shown to progress to an adaptive T-cell
response characterized by T-cell proliferation, and secretion of
T-cell cytokines. Investigation of the ability of protein aggregates
to induce cytokine signatures as biomarkers of immune responses
is essential for determining their risk of immunogenicity.
BIOT 357 – 10:10 a.m.
Beta-amyloid and transthyretin: is there a natural antiAlzheimer’s drug?
Jiali Du, Patricia Cho, Dennis Yang, Regina M. Murphy, regina@engr.
wisc.edu.Department of Chemical and Biological Engineering, University of
Wisconsin, Madison, WI 53706, United States
Alzheimer’s disease is a devastating neurodegenerative disorder
that afflicts over 5 million people in the US. One of the dominant
pathological features of the disease is the deposition of beta-amyloid
as fibrillar aggregates, in extracellular amyloid plaques. According
to the widely held ‘amyloid cascade’ hypothesis, aggregation of
beta-amyloid is causally linked to neuronal dysfunction and death.
Beta-amyloid is a 4kDa peptide clipped from a precursor protein,
APP. In an effort to develop an animal model of Alzheimer’s disease,
researchers have generated transgenic mice that express APP. The
mice exhibit extensive beta-amyloid deposits, but surprisingly
there is little neurotoxicity. Recently, it has been demonstrated that
the transgenic mice upregulate expression of transthyretin (TTR),
and the data strongly suggests that TTR protects the mouse neuron
from beta-amyloid toxicity. These results raise several questions:
how does TTR exert its protective effect? Is it lost in Alzheimer’s
patients? Can it be restored? In this presentation we will describe
our efforts to answer these questions. We have used LC/MS/MS,
peptide arrays, and scanning alanine mutagenesis to determine
the specific residues of TTR that are involved in binding to betaamyloid. Strikingly, we have observed a strong correlation between
increased binding to beta-amyloid, and reduced quaternary
stability of TTR. We are characterizing in detail the effect of
TTR (wt and mutants) on beta-amyloid aggregation. Finally, in
collaboration with Dr. Jeff Johnson, we are examining the effect
of various TTR mutants on beta-amyloid toxicity, in an effort to
determine the features of TTR that regulate beta-amyloid toxicity.
BIOT 358 – 10:50 a.m.
Hofmeister effects on Sup35NM aggregation kinetics, amyloid
thermostability and strength
Jonathan Rubin1, [email protected], Andreas S. Bommarius1,3,
Sven H. Behrens1, Yury O. Chernoff2. (1) Chemical and Biomolecular
Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332,
United States (2) Biology, Georgia Institute of Technology, Atlanta, Georgia
30332, United States (3) Chemistry and Biochemistry, Georgia Institute of
Wednesday Morning
BIOT 355 – 9:10 a.m.
Technology, Atlanta, Georgia 30332, United States
Prion proteins are capable of converting from their soluble,
biologically-functional forms into highly ordered, fibrous cross-β
aggregates, known as amyloids. This conversion is associated
with certain neurodegenerative conditions, such as Alzheimer’s
disease. Protein misfolding is strongly influenced by specific ionic
effect, known as Hofmeister effects. Using a yeast prion protein,
Sup35NM, we tested the influence of a host of sodium salts on
aggregation kinetics; namely the salts’ effect on elongation rate and
lag time. Salts known to destabilize protein, chaotropes, exhibited
long lag times and slower elongation rate; whereas salts known to
stabilize proteins, kosmotropes, showed short lag times and fast
elongation rates. Thermostability of the resulting aggregates was
then studied using DLS and gel electrophoresis. Chaotropes were
significantly more thermostable than kosmotropes. The amyloids
formed in different salts were also transfected in yeast and tested
for their “strength.” With stability, kinetic and strength data, a.
aggregation mechanism is postulated.
BIOT 359 – 11:10 a.m.
Xanthene food dye: A novel modulator of amyloid-beta
peptide aggregation and the associated impaired neuronal cell
function
H. Edward Wong, Inchan Kwon, [email protected] of
Chemical Engineering, University of Virginia, Charlottesville, Virginia
22904, United States
Alzheimer’s disease (AD) is the most common form of dementia.
It has been widely accepted that aggregation of amyloid-beta
peptide (Abeta) is causative to the onset of AD. In the search for
safe, effective modulators, we evaluated the modulating capabilities
of erythrosine B (ER), a Food and Drug Administration (FDA)approved red food dye, on Abeta aggregation and cellular
responses. Biophysical and immunological studies showed that
protofibrils are predominantly generated and stabilized, and length
of the ER-induced protofibrils is inversely proportional to the ER
concentration. Our findings show that ER is a novel modulator
of Abeta aggregation and reduces Abeta-associated impaired cell
function.
135
12:30 -2:00 p.m.
2:00 p.m.
A
B
D
E
Room# 6A
Program Chair’s Meeting
Room# 16A
BIOT 360 – 2:00 p.m.
2:00 p.m Room# 16B
Upstream Processes: Protein Engineering – Methods and Applications
A.Link, A. Rakestraw Papers 368-375
2:00 p.m.
Room# 25A S. Vunnum, S. Ahuja Papers 376-382
136
2:00 p.m.
Room# 16A
J. Vogel, J. Hubbuch, M. Ottens, R. Willson,
O. Thomas Papers 360-367
J. Vogel, J. Hubbuch, M. Ottens, R. Willson, O. Thomas Papers 360-367
Advances in Biotechnology Product Development: Predictive Modeling,
Small-Scale Modeling, and High-Throughput Process Development
Accelerating Downstream Development
and Process Transfer
Downstream Processes: Accelerating Downstream Development and
Process Transfer
Downstream Processes:
2:00 p.m. Room# 25B
Biophysical & Biomolecular Processes: High Concentration Protein
Therapeutics -Development, Production, and Delivery
J. Maynard, H. Samra Papers 383-389
6:00 p.m. Room# Hall E
Reception/Poster Session S. Singh, C. Collins Various Paper Numbers
Statistical vs. stochastic experimental design: An experimental
comparison based on the example of protein refolding
Bernd Anselment, [email protected], Dirk Weuster-Botz.
Institute of Biochemical Engineering, Technische Universität München,
Garching, Germany
Optimization of experimental problems is a challenging task
in both engineering and science. In principle two different
experimental design strategies exist: statistical and stochastic
(heuristic) methods. Both aim to efficiently and precisely identify
optimal solutions inside the problem specific search space. Here
we evaluate both strategies on the same experimental problem,
the optimization of the refolding conditions of the lipase from
Thermomyces lanuginosus with 26 variables under study.
We compared the following methods: First, a statistical design
consisting of a D-optimal screening step followed by an
optimization via response surface methodology. Second, an
iterative stochastic optimization applying a genetic algorithm. Both
methods were experimentally evaluated several times to account
for the stochastic beginning of the genetic algorithm and analyze
changes in the variable setup of the statistical design. Interestingly,
the results show that only the stochastic optimization was able to
identify the global optimum (~1.400 U∙g-1 refolded activity, 6-fold
higher than the reference) correctly.
BIOT 361 – 2:20 p.m.
High-throughput acquisition of physicochemical parameters
from crude feedstocks for model-based protein purification
process development
in Life Sciences, Karlsruhe Institute of Technology, Karlsruhe, Germany
Today’s competitive biopharmaceutical market requires short
process development times, while regulatory agencies require
stringent product quality and safety requirements to be upheld. To
meet these seemingly opposing needs the industry still relies on
empirically developed platform process designs, often resulting in
suboptimal process efficiency.
The FDA’s ‘Quality by Design’ initiative encourages the use of novel
design tools for process development, such as a hybrid approach
utilizing High-Throughput Experimentation to acquire the
physical-chemical and thermodynamic data necessary for modelbased in-silico purification process development.
Gradient Chromatofocusing has been proven to be an efficient
tool for high-resolution separation of crude feedstocks and a
compounds elution-pH a powerful parameter for the design of
ion-exchange chromatography unit operations. We present how
linear pH-gradients can be utilized as a flexible first separation
dimension in a multidimensional fractionation scheme designed to
deliver many of the parameters necessary for process development
from small amounts of crude feedstock.
BIOT 362 – 2:40 p.m.
Introducing single domain antibody based affinity ligands for
antibody detection and characterization in label free binding
platforms
Pim Hermans, [email protected]&D, BAC BV, Leiden, Holland
2333 CH, The Netherlands
The use of bacterial surface proteins like Protein A and L can be highly
effective for the purification and detection of antibodies. However,
there are gaps in the functionality that they provide, and when a
target protein falls into one of these gaps, the process of finding an
alternative strategy can be extremely frustrating. For this reason
BAC has developed a range of “CaptureSelect” affinity ligands,
derived from heavy chain antibodies found in camelids, directed
against a unique panel of antibody sub-domains, like IgG-CH1, CH2, - CH3, kappa - and lambda light chains. When conjugated
to biotin, excellent compatibility with different streptavidin
based biosensors is demonstrated providing researchers valuable
tools for label free detection, quantitation and characterization
of in principle any antibody format (e.g. Fab fragments). Novel
analytical concepts will be described that accelerate downstream
process development and enable monitoring of product quality
during manufacturing.
Alexander T. Hanke1, [email protected], Frieder Kröner2, Beckley
K. Nfor1, Martijn W.H. Pinkse1, Peter D.E.M. Verhaert1, Luuk A.M.
van der Wielen1, Jürgen Hubbuch2, Marcel Ottens1. (1) Department of
Biotechnology, TU Delft, Delft, The Netherlands (2) Institute of Engineering
137
Wednesday Afternoon
Wednesday
Afternoon
Wednesday Afternoon Sessions
Hybrid experimental simplex algorithm, a novel optimization
method for high throughput experimentation: Case studies in
ion exchange chromatography
Spyridon Konstantinidis1, [email protected], Sunil
purification process at microtiterplates toward labscale columns
limited in cell culture material. The examples presented will show
the benefits of the smart developments in microfluidics and HTS.
BIOT 365 -4:00 p.m.
Chhatre1, Eva Heldnin2, Nigel Titchener-Hooker1. (1) Department of
Biochemical Engineering, University College London, London, United
Kingdom (2) Biotechnologies R&D, GE Healthcare Life Sciences, Uppsala,
Optimization of multiproduct antibody facility designs: A
genetic algorithm approach
Sweden
Ana S. Simaria1, [email protected], Ying Gao2, Richard Turner2,
High throughput screening during the early-stage development
of bioprocesses frequently involves the generation of statistical
designs to investigate the experimental spaces of interest. However,
the information gained by such designs may be outweighed by
the analytical burden so-created. The employment of the Simplex
Algorithm to select a subset of test conditions to evaluate from
a search space has been shown previously to be beneficial in
countering this challenge. This paper augments the established
Simplex method to form a novel Hybrid Experimental Simplex
Algorithm which has a superior capability of identifying optimal
experimental conditions in search spaces which are mapped by
a defined grid of test conditions. The paper describes the new
algorithm and illustrates its use in two case studies conducted in a
96-well filter plate format where it is shown to converge consistently
to optimal locations while reducing significantly the total number
of experimental conditions needed to be tested.
BIOT 364 – 3:40 p.m.
High throughput screening as a tool for developing purification
processes
Michel Eppink, [email protected] Processing,
Synthon BV, Nijmegen, Gelderland 6503GN, The Netherlands
“Time to market” is for the biotechnology industry an important
milestone to improve overall efficiency and reduce cost price.
A crucial part of the biotechnology processes concerns the
development of the downstream process (DSP) for therapeutic
proteins (biologicals). In this way fast methods such as high
throughput screening techniques and microfluidics are needed to
speed up DSP processes.
In this presentation smart developments in microfluidics and
high througput screening (HTS) technologies will be discussed as
very efficient tools in designing/predicting/scaling of purification
processes. Normally, limited amounts of cell culture material
is present during the development of a purification process for
a specific biological. How are we able not only to get a detailed
understanding of the biological as well as translating the developed
138
Suzanne S. Farid1. (1) Department of Biochemical Engineering, University
College London, London, London WC1E 7JE, United Kingdom (2)
MedImmune Limited, Cambridge, United Kingdom
Increasing pressures exist in the biopharmaceutical sector for the
design of flexible and cost-effective multi-product facilities that
can cope with diverse drug candidate characteristics and process
variations. This is due to the high risks of clinical failure as well
as pressures to contain costs and enhance capacity utilisation. To
address this problem, a genetic algorithm is created that can handle
multiple decisions, trade-offs and uncertainties simultaneously
to design multi-product facilities, where the decision variables
represent choices at different levels (facility, product, sequence,
unit operation). An industrially-relevant case study is presented
that addresses the design of commercial manufacturing facilities
for the production of monoclonal antibodies. The most costeffective purification sequences and equipment sizing strategies
that meet demand and purity targets are identified for each product
and methods to visualise trade-offs in the set of optimal solutions
with similar cost values are provided so as to enhance the decision
making process.
BIOT 366 – 4:20 p.m.
Mechanism of interaction between proteins and the mixedmode adsorbent, Capto MMC
Nils Wallménius, Enrique Carredano, Karol M. Lacki, karol.lacki@
ge.com, Eggert Brekkan.Biotechnologies, GE Healthcare Lifesciences,
Uppsala, SE 75184, Sweden
Capto™ MMC is a mixed-mode chromatographic adsorbent that
is functionalized with a ligand carrying both a hydrophobic and
an electrostatic group. The dual functionality makes optimization
of conditions for binding and elution more challenging. Better
understanding of the interaction between proteins and the
mixed-mode adsorbent is needed in order to facilitate rapid
development/evaluation of a chromatographic step utilizing the
unique properties of the adsorbent. Thermodynamic modeling of
protein adsorption on Capto MMC is done by utilizing established
adsorption isotherm. Data are obtained through automated robotic
high-throughput screening in 96-well microtitre plates and show
the effect of salt concentration and pH, and influence of the ligand
hydrophobicity under the different conditions, on the interaction
of model proteins and a monoclonal antibody with Capto MMC.
In addition, experimental designs based on adsorption isotherm
models and designs used in standard DoE studies are compared
and their applicability in studies describing adsorption equilibria
are discussed.
BIOT 367 – 4:40 p.m.
Simple and fast methods for predicting the dynamic binding
capacity by using 96-well format micro-plates and small
chromatography columns
Shuichi Yamamoto, [email protected], Yuki Suehiro,
Hiroyuki Nagaoka, Noriko Yoshimoto, Kazunobu Minakuchi.BioProcess Engineering Laboratory, Yamaguchi University, Tokiwadai, Ube,
Yamaguchi 755-8611, Japan
Chromatography is the main purification unit operation for
biopharmaceuticals. However, there are so many variables
or parameters such as flow-rate, gradient slope and column
dimension to be tuned to find the optimized operating and/
or chromatography conditions. The mobile phase must also be
carefully examined as the separation and/or the binding capacity
is strongly influenced. For this reason fully automated (robotic)
systems using a 96-well micro-plate format have been developed
by many researchers as high-throughput process development
methods for chromatography of proteins. However, methods for
predicting the process-scale column performance based on the
information obtained with the micro-plate system have not yet
been fully developed.
In this study, we carried out the batch adsorption experiment (IgGprotein A system) by using the 96-well micro-plate for determining
the adsorption isotherm. In most cases the working liquid volume
was 0.1-0.2 mL and the gel volume was 0.004-0.010 mL. The
dynamic binding capacity (DBC) values were measured as a
function of residence time with a small column of ca. 1 mL (5 mm
id x 50 mm). For rigid and small silica based resins it was possible
to pack a smaller column of ca. 0.3 mL (5 mm id x 15 mm), which
shows the same DBC values with the 1 mL column.
A simple method for predicting the dynamic binding capacity
(DBC) was proposed, which uses a correlation between the DBC/
SBC (SBC: static binding capacity) and the normalized residence
time with the particle diameter and the pore diffusivity. In this
method the void fraction was considered in order to use the data
for scale-up studies. The experimental data were well-correlated
based on this correlation equation.
Upstream Processes:
Protein Engineering – Methods and
Applications
2:00 p.m Room# 16B
A.Link, A. Rakestraw Papers 368-375
BIOT 368 – 2:00 p.m.
Evolution of customized regulatory proteins for use as in vivo
molecular reporters
Joseph A Gredell, [email protected], Christopher S Frei, Patrick C
Cirino.Department of Chemical and Biomolecular Engineering, University
of Houston, Houston, TX 77096, United States
Microbial small-molecule biosensors and endogenous molecular
reporters are utilized in a variety of applications that include highthroughput screening of biosynthesis libraries, environmental
monitoring, and novel gene regulation in synthetic biology.
Regulatory proteins directly couple effector recognition with gene
transcription and are ideally suited to these applications, but their
utility is limited to the range of native effector molecule(s) to which
they respond.
Our laboratory has previously established that the specificity
of the E. coli AraC regulatory protein can be altered from the
natural ligand L-arabinose to D-arabinose as well as to the more
industrially relevant compounds mevalonate and triacetic acid
lactone. This presentation will 1) describe several additional AraC
variants engineered to respond to new target metabolites, and 2)
outline our understanding of AraC specificity evolution pathways
as they relate to different FACS-based dual screening strategies.
BIOT 369 – 2:20 p.m.
Design of a hydrophobically-enhanced protein nanocapsule for
molecular encapsulation and transport
Dongmei Ren1, [email protected], Mercè Dalmau1, Arlo Z Randall2,3,
Pierre F Baldi2,3, Matthew M Shindel1, Szu-Wen Wang1. (1) Department
of Chemical Engineering and Materials Science, University of California,
Irvine, Irvine, CA 92697, United States (2) School of Information and
Computer Sciences, University of California, Irvine, Irvine, CA 92697,
United States (3) Institute for Genomics and Bioinformatics, University of
California, Irvine, Irvine, CA 92697, United States
Encapsulation and delivery of antitumor therapeutics is one of the
challenges in targeted drug delivery. We have exploited a 25-nm
dodecahedron protein nanoparticle from the E2 subunit of pyruvate
139
Wednesday Afternoon
BIOT 363 – 3:00 p.m.
BIOT 370 – 2:40 p.m.
Evolution of thermo stable fluorescent proteins using protein
engineering and ratio metric sorting
Patricia Langan1, [email protected], Csaba Kiss2, Devin Close2,
Andrey Kovalevsky2, Geoff Waldo2, Claire Sanders2, Kassidy Burnett2,
Babs Marrone2, James Freyer2,3, Andrew Bradbury2. (1) University
of New Mexico and Los Alamos National Lab, Biomedical Engineering
and Bioscience, United States (2) Los Alamos National Lab, Bioscience,
Los Alamos, NM 87545, United States (3) University of New Mexico,
Biomedical Engineering, Albuquerque, NM 87131, United States
The development of Fluorescent Proteins as reporters and labels
has aided the way complex biological systems can be visualized and
interrogated. However, the broad application of most fluorescent
proteins has been limited by their thermal and pH sensitivity.
We have evolved an extremely thermo-stable fluorescent protein.
This protein is being used as a template to carry out site directed
mutagenesis and molecular evolution to modify the emission
wavelength from the green towards the red. Amino acids that could
affect the dynamics of the fluorophore were chosen for mutation.
A unique aspect of this work is our use of the National Flow
Cytometry Resource in order to rapidly analyze and sort colorshifted clones. In addition to red-shifted clones, other interesting
phenotypes were obtained, including an array of photo-sensitive
fluorescent proteins. The results obtained using this innovative
approach, and their implications for developing a robust palette of
thermo-stable fluorescent proteins were realized.
140
BIOT 371 – 3:00 p.m.
Engineered allosteric switches for sensing peptides
Glenna Meister1,2, [email protected], Neel Joshi1,2.
(1) School of Engineering and Applied Science, Harvard University,
Cambridge, Massachusetts 02138, United States (2) Wyss Institute for
Biologically Inspired Engineering, Boston, Massachusetts 02115, United
States
Allosteric proteins undergo a conformation change upon binding
of a substrate that modulates their activity at a distal site. These
proteins could be extremely useful as biosensors, however, few
naturally occurring allosteric proteins are capable of binding
to a target of interest and producing an easily detectible signal.
Engineered allosteric switches can be created by fusing two
independent domains—an “input” domain that binds the target,
and an “output” domain, that is actuated by the first domain. We
hypothesized that the calcium-binding protein, calmodulin, is an
ideal input domain due to its large conformational changes upon
binding a peptide. We created fusions of calmodulin to fragments of
the enzyme TEM1 beta-lactamase varying the locations and linker
lengths of the fusion points. We were able to confirm switching
activity in some fusion constructs, and one fusion enzyme had a
33-fold increase in activity when bound to the calmodulin-binding
peptide, mastoparan.
BIOT 372 – 3:40 p.m.
Novel model for understanding mechanical forces of enzyme
and its application to enhance enzyme activity and stability
So Yeon Hong1, [email protected], Jeong Chan Joo2, Young Je Yoo1,2.
(1) Graduate Program of Bioengineering, Seoul National University, Seul,
none 151-742, Republic of Korea (2) School of Chemical and Biological
Engineering, Seoul National University, Seoul, none 151-742, Republic of
Korea
Under the induced fit hypothesis, enzyme structure is changed
to fit substrate and is recovered to its original form after catalysis
occurred. This phenomenon resembles elastic body motion so
that spring model is proposed to analyze mechanical change of
enzyme. This modeling method uses 2 forms of pdb files, apo and
holo form of enzyme structure, to analyze the forces by substrate
binding. Validation was performed from the data of bacteriophage
T4 lysozyme, HIV-1 protease and Candida antarctica lipase B.
Spring model was used with the aim of activity enhancement
for the selection of target mutation sites. It was discovered that
some mutations showed enhancement of activity at the forced
region in bacteriophage T4 lysozyme. The mutants of Candida
antarctica lipase B showed about 4 times enhanced activity when
flexible amino acids were introduced into the forced region. In the
saturation mutagenesis of the residue having the highest torque
value using Bacillus circulans xylanase, it was shown that activity
was increased about 1.6 times without stability loss. The details will
be explained and discussed.
BIOT 373 – 4:00 p.m.
Exploiting the biophysical properties of Centyrins for biologics
drug development
Steven Jacobs, [email protected] Division of Johnson & Johnson
Pharmaceutical Research & Development, L.L.C., Centocor Research &
Development, United States
Alternative scaffolds represent an emerging class of protein
therapeutics that combine the attractive specificity properties of
mAbs with the simplicity, ease of manufacture and tissue penetration
associated with small molecules. One such scaffold, the Fibronectin
type III (FN3) domain is often observed in natural protein-protein
interactions. These domains have significant structural homology
to the Ig domains found in antibodies with loops that are analogous
to CDRs. Working from the premise that sequence conservation
arises from evolutionary pressure to maintain stability elements,
we have designed novel consensus FN3 domains, called Centyrins.
One such Centyrin (Tencon) is derived from a sequence alignment
of 15 FN3 domains from human tenascin. Tencon has high
thermal stability, excellent solubility, and exhibits high levels of
soluble expression in E. coli. X-ray crystallographic studies have
confirmed that this novel protein scaffold does indeed fold into a
FN3 type structure as designed. We are exploiting the properties of
this exceptionally stable scaffold to develop a series of molecules
aimed at broadening the therapeutic applications of biologics to
areas such as bispecific drugs, intracellular inhibitors, and for
use in alternative routes of drug delivery. The development and
application of the Centyrin technology including novel methods
of library design will be presented together with in vitro and in
vivo characterization of high-affinity Centyrins against an array of
therapeutic targets.
BIOT 374 – 4:20 p.m.
De novo design of protein binders: Targeting human IgG (Fc)
Eva-Maria Strauch1, [email protected], Sarel J Fleishman1,2, David
Baker1. (1) Biochemistry, University of Washington, Seattle, Washington
98195, United States (2) Department of Biological Chemistry, Weizmann
Institute of Science, Rehovot, Israel
protein-interaction networks. We recently developed a general
computational method for the generation of de novo protein
binders. Our methods focus on the incorporation of “hot-spot”
residues onto protein scaffolds to generate protein binders to
specific epitopes on proteins of interest. We demonstrate how
combinations of a small number of newly generated high-affinity
sidechain interactions with the target epitope of human IgG result
in a new set of Fc binding proteins with new characteristics. By
applying directed evolution methodologies via yeast surface
display and deep sequencing, we optimized successful designs
and explored their propensity for new properties, such as pH
dependence and specificity for antibodies of different species. Our
computationally designed and evolved proteins will have direct
practical applications, such as providing new material for affinity
chromatography for the purification of various antibodies or Fcfusion proteins.
BIOT 375 – 4:40 p.m.
Using directed molecular evolution to understand and improve
molecular recognition in novel scaffold affinity reagents
Brenda M Beech3, Dayle MA Smith2, Thomas C Squier1, Cheryl L
Baird1, [email protected]. (1) Department of Cell Biology and
Biochemistry, Pacific Northwest National Laboratory, Richland, WA
99352, United States (2) Department of Computational Biology and
Bioinformatics, Pacific Northwest National Laboratory, Richland, WA
99352, United States (3) College of Sciences, Washington State University,
Richland, WA 99352, United States
Our ultimate goal is to develop a rapid means to create nonantibody-based affinity reagents. Our strategy involves using
directed molecular evolution and yeast surface display as a tool to
generate panels of variant proteins that bind a defined ligand with
improved affinity compared to the native protein. Through the
analysis of these variants, we gain an understanding of the drivers
that govern molecular recognition such that computational models
for the rational design of new binding clefts can be developed. Our
model affinity scaffold is based on calmodulin, a naturally occurring
bivalent protein with considerable molecular plasticity and high
structural stability. We have identified mutants with enhanced
binding affinities to natural peptide targets that in all cases involve
mutations far from the binding interface. Specifically, a range of
mutations that either destabilize salt bridges in the unbound state
or stabilize salt bridges in the bound state enhance binding. These
results suggest that simple energetic calculations based on changes
in the free energies of proteins provides a predictive means for
rationale protein engineering.
The ability to design highly specific protein binders to disrupt
protein-protein interactions would have immediate application
to treatment of many diseases and for the dissection of
141
Wednesday Afternoon
dehydrogenase to develop a universal strategy for encapsulating
drug molecules through only non-covalent interactions. To
enhance molecular encapsulation capacity for the model antitumor
drug doxorubicin, a biomimetic hydrophobic microenvironment
was engineered within the E2 scaffold cavity by mutating native
hydrophilic residues to phenylalanines. The mutation sites were
selected based on molecular structure modeling and computational
analysis. The resultant scaffold exhibited over a 35-fold increase in
drug encapsulation relative to native E2 scaffold, yielding high drug
loading levels compared to conventional nanoparticle delivery
systems. Doxorubicin-loaded E2 nano-capsule demonstrated
significant cellular uptake. Intracellular release was shown by
cytotoxicity against breast cancer cells, with an IC50 of 0.33 ± 0.12
mM. Our novel protein engineering approach shows the promise
of utilizing hydrophobic interactions to fabricate protein nanocapsule for molecular encapsulation and transport.
Predictive Modeling, Small-Scale Modeling,
and High-Throughput Process Development
2:00 p.m.
Room# 25A S. Vunnum, S. Ahuja Papers 376-382
BIOT 376 – 2:00 p.m.
Considerations for developing a well-characterized bioreactor
scale-down model
LiYing Yang, [email protected], David Lindsay, Mei Shao,
Brian Stamper.Manufacturing Sciences and Technology, MedImmune,
Frederick, Maryland 21703, United States
Bioreactor scale-down model demonstration and qualification is a
key component in process development, process characterization,
manufacturing support / troubleshooting and continuous
improvement. 4-Liter bench-top bioreactor models were designed
and developed as a scale-down model of the 15,000-Liter
commercial scale bioreactor. Considerations on bioreactor
design, scale-up and scale-down strategy will be discussed. Mass
transfer characterization studies were conducted, from which
the results were summarized and utilized to guide scale-down
model development. Results from multiple fed-batch cell culture
processes will be discussed to demonstrate the suitability of the 4-L
scale down model.
BIOT 377 – 2:20 p.m.
Iterating to a solution: Refinement of a small-scale bioreactor
model
Pratik Jaluria, [email protected], E. Todd Sorensen, John Facenda,
Hunter Malanson, Anne Kantardjieff.Department of Upstream
Development, Alexion Pharmaceuticals, Cheshire, CT 06410, United States
Motivated by the need to reliably predict large-scale, cell culture
performance for the production of recombinant proteins and
monoclonal antibodies, we set out to refine our small-scale
bioreactor model. Our approach started by defining requirements
for our model such as retaining product quality attributes across
scales and matching trends in growth and productivity. Next,
142
we employed computational fluid dynamics (CFD) software in
conjunction with a series of experiments to characterize each
scale, focusing on: magnitude of shear forces, mixing time, bubble
size distribution and liquid addition dispersion. We explored the
impact of modifying a host of operating conditions including:
agitation rate, pH setpoint, dissolved oxygen setpoint, sparge rate,
location of liquid additions (i.e. feed and alkaline solution) and
timing of liquid additions. Ultimately, we engineered our model
to go from outperforming the large-scale by nearly 50% in terms
of productivity to closely matching final titer while preserving
product quality.
BIOT 379 – 3:00 p.m.
BIOT 378 – 2:40 p.m.
21250, United States
High-throughput clonal selection of antibody-producing CHO
cells using a microfluidic cell culture platform
Véronique Lecault1,2,3, [email protected], William J
Bowden1,4, Anjali Verma5, Anupam Singhal2,3, Joe Orlando5,
Christopher Martin5, James M Piret1,3, Carl L Hansen2,4. (1) Michael
Smith Laboratories, University of British Columbia, Vancouver, BC V6T
1Z4, Canada (2) Centre for High-Throughput Biology, University of British
Columbia, Vancouver, BC V6T 1Z4, Canada (3) Department of Chemical
and Biological Engineering, University of British Columbia, Vancouver, BC
V6T 1Z3, Canada (4) Department of Physics and Astronomy, University
of British Columbia, Vancouver, BC V6T 1Z1, Canada (5) EMD Millipore,
Bedford, MA 01730, United States
Mammalian cells are widely used for the production of
recombinant proteins for research, diagnostics, and biologic
pharmaceuticals. The clonal selection of high-producing cells is
a bottleneck in the development of new production processes.
Current techniques for the selection of clones are time-consuming,
labour-intensive and costly. We present a microfluidic platform
for clonal analysis that provides combined measurements of
cell-specific protein production and proliferation using Chinese
Hamster Ovary (CHO) suspension cells. This cell culture array
technology can screen hundreds of single cells simultaneously in
nanovolume chambers. An immunocapture bead assay in each
4.1 nanoliter culture chamber rapidly identifies highly productive
cells. We show that the high effective concentration of a single
cell inoculum in a nanoliter volume chamber (2.4 x 105 cell/ml)
provides an enhanced cell culture environment and higher plating
efficiency. This technology has been applied to the clonal selection
of monoclonal antibody-producing CHO cells.
Comparability of monoclonal antibody titers and its
glycosylation profile produced in minibioreactors vs. bench
scale bioreactors
Shaunak D Uplekar1,3, [email protected], Kurt A Brorson2,
William R LaCourse3, Antonio R Moreira1, Govind Rao1. (1) Center for
Advanced Sensor Technology, Department of Chemical and Biochemical
Engineering, University of Maryland Baltimore County, Baltimore, MD
21250, United States (2) Division of Monoclonal Antibodies, Center for
Drug Evaluation and Research, Food and Drug Administration, Silver
Spring, MD 20903, United States (3) Department of Chemistry and
Biochemistry, University of Maryland Baltimore County, Baltimore, MD
QbD and PAT regulatory initiatives aim to achieve consistent
production of product having predefined quality and demand
numerous process development studies to establish design
space for each product quality attribute. Novel high-throughput
minibioreactors have a great potential to fulfill this demand.
However, their comparability with conventional bench scale
systems needs to be established. Comparability studies of highthroughput minibioreactors versus 5L bioreactors focusing on
gene profiling have been discussed in the past. Yet, protein titer and
quality aspect has not been dealt with. Here, we further investigate
the comparability by looking into titers and N-glycan profiles of
monoclonal antibody produced by serum free mammalian cell
culture. We use a two-step scheme to purify IgG3 antibody from
the cell culture broth and carry out N-glycan analysis using high
pH anion exchange chromatography with pulsed amperometric
detection. Influence of pCO2 in minibioreactors and bench scale
systems is also discussed.
BIOT 380 – 3:40 p.m.
Integrated analytical tools and screening platforms for HTPD
as well as modern electrophoresis and chromatography technologies
are combined with DoE approaches from BI’s RAPPTor® automated
screening platform. Together with QbD aspects this strategy
delivers fully scaleable, well understood and robust processes. The
benefit of the seamless combination of analytical tools with highly
automated high throughput technologies will be presented in a
case study and some examples.
BIOT 381 – 4:00 p.m.
High throughput process development: Identifying key
operating parameters for retrovirus clearance by AEX and
mixed mode resins
Lisa Connell-Crowley1, [email protected], Elizabeth
Larimore2, Ron Gillespie1, Suresh Vunnum1. (1) Process & Product
Development, Amgen Inc.,, Seattle, WA 98006, United States (2) University
of Washington, Seattle, WA, United States
Virus contamination is a serious concern for biotherapeutics
produced in mammalian cells. Regulatory agencies require that
downstream processes demonstrate clearance of viruses and
virus-like particles as part of a multi-pronged approach to viral
safety. Recent studies have used Design of Experiment (DOE)
strategies on columns to define design spaces for virus removal
on chromatography resins, however these types of studies can
use significant amounts of material, virus and time. To reduce the
time and material needed for defining key parameters, we utilized
a high-throughput screening approach in a plate-based batch
binding format to examine retrovirus clearance on resins used in
flowthrough mode for monoclonal antibody (mAb) purification.
The results provide a broad picture of the impact of pH, salt strength,
mAb binding, and impurity binding on the retention of xMuLV on
Q Sepharose® Fast Flow and Captoadhere® resins and are consistent
with results obtained using a column format. This approach can be
used to provide valuable information during process development
to ensure that the process meets viral clearance targets.
Philine Dobberthien, philine.dobberthien@boehringer-ingelheim.
com, Joey Studts.Purification Development, BioPharmaceuticals,
Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss,
Biberach an der Riss 88397, Germany
Fast process development, process knowledge and lean development
are no contradictions. Protein Science at Boehringer Ingelheim has
developed the BI-PurEx strategy with integrated analytical tools
that allow early decision making and support most efficient process
and formulation development while gaining process knowledge
and understanding.
In very early phases with limited material availability miniaturized
high-throughput analytics with biophysical and biochemical assays
including thermal stability, virial coefficient and impurity detection
BIOT 382 – 4:20 p.m.
High throughput process development of a commercial second
generation antigen purification process
Doug MacDonald1, [email protected], Randy Ng1,
Richard Blackmore1, John Zeng1, Brian Sims-Fahey2, Eva Aw2. (1)
Protein Process Development, Dendreon, Seattle, WA 98121, United States
(2) Analytical Development, Dendreon, Seattle, WA 98121, United States
The commercial purification process for an FDA approved insect
cell-derived Cellular Immunotherapy was re-developed. The
goal of the second generation process was to reduce the number
143
Wednesday Afternoon
Advances in Biotechnology
Product Development:
BIOT 382.1 – 4:40 p.m.
Biophysical & Biomolecular
Processes:
High Concentration Protein Therapeutics Development, Production, and Delivery
2:00 p.m. Room# 25B
J. Maynard, H. Samra Papers 383-389
BIOT 383 – 2:00 p.m.
AAPH induced mAb oxidation and aggregation
Characterizing the viscosity behavior of concentrated protein
coformulations
Kai Zheng1, [email protected], Diya Ren2, Wayne Lilyestrom1,
William J Galush, [email protected], Mansij S Hans, Lan N
Yatin Gokarn1, Robert Bayer2, Thomas Scherer1, Y. John Wang1, Junyan
A. Ji1. (1) Late Stage Pharmaceutical Development, Genentech, South
San Francisco, California 94080, United States (2) Oceanside Pharma
Technical Development, Genentech, Oceanside, California 92056, United
States
Oxidation is a key degradation pathway in protein drugs. During
a therapeutic antibody formulation development, oxidation
stress was induced by a free radical generator, 2,2’-Azobis
(2-amidinopropane) dihydrochloride (AAPH). In addition to
methionine and tryptophan oxidation, we observed the aggregation
increase. Size-exclusion chromatography and multi-angle light
scattering (SEC-MALS) showed that high molecular weight species
(HMWS) contained dimer, tetramer, and higher order aggregates.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) showed that the newly formed HMWS was mainly
covalently linked and intermolecular disulfide cross-linking was
a major contributing factor. Tryptic map indicated methionine
and tryptophan oxidation, but did not reveal any non-disulfide
cross-linking sites. Fluorescence spectra suggested that bityrosine
may contribute to cross-links. Additional study indicated that
tryptophan, tyrosine, or pyridoxine could protect antibodies’
aggregate formation from AAPH stress.
In summary, our data exhibit the complicated mAb oxidation
products under chemical stress and mAb oxidation should be
closely monitored during biotherapeutic development.
Le, Jamie MR Moore.Early Stage Pharmaceutical Development, Genentech,
Inc., South San Francisco, CA 94080, United States
High concentration protein products are becoming increasingly
commonplace within the biopharmaceutical industry, driven by
the need for high doses of subcutaneously administered drugs. An
undeveloped variant of these products features multiple proteins
coformulated together in high concentration protein mixtures.
Previous work has illustrated that the viscosity of aqueous
solutions of two highly similar proteins can be remarkably different
at a specific concentration. This work characterizes the viscosity
behavior of mixtures of such proteins, primarily monoclonal
antibodies, and shows that a simple mixing rule predicts the
viscosity of an arbitrary, coformulated mixture of proteins across
different proportions, concentrations, and ionic strengths. Only
information about the viscosity of the proteins by themselves is
required. Extensions of this analysis may provide further insight
into the interactions at play in highly concentrated protein
solutions.
BIOT 384 – 2:20 p.m.
Stabilization of proteins at high concentrations by crowding
Jennifer A Maynard, [email protected], Keith P Johnston,
Tom M Truskett, Aileen Dinen, Ameya Bornwankar, Brian K Wilson, Tarik
Khan, Kevin Kaczorowski.Chemical Engineering, University of Texas at
Austin, Austin, TX 78705, United States
Protein stability at high concentrations (200-400 mg/ml) is of broad
interest in science and human health. In cellular environments, a
variety of macromolecules crowd proteins into in a compact state,
stabilizing folded proteins, as described theoretically and observed
144
experimentally. However, short-ranged attractive and other
interactions between closely spaced proteins and model colloids
often produce irreversible aggregation and precipitation. In
vitro, these undesirable events dominate, thus novel concepts are
needed to mimic nature successfully to stabilize proteins at high
concentration. We have created concentrated antibody protein
dispersions, in the form of dense 80-400 nm clusters of protein
molecules, which are conformationally stabilized by crowding and
retain therapeutic activity in vivo. The size of the relatively large,
equilibrium nanoclusters is tuned by addition of macromolecular
crowding molecules, which provides strong short-range attractions
to balance weak longer-range electrostatic repulsions between
individual protein molecules near the isoelectric point. Whereas
the locally concentrated environment within nanoclusters stabilizes
the native state of protein molecules through self-crowding,
weak interactions between nanoclusters result in colloidallystable, translucent dispersions with low viscosities. Not only is
the protein native structure maintained within the nanocluster,
but upon dilution, the clusters reversibly dissociate into native
monomeric protein molecules with high biological activity. The
viscosities of antibody nanocluster dispersions are sufficiently
low to allow subcutaneous injection to mice at typical therapeutic
antibody dosages. Administration of dispersions resulted in high
bioavailability and biological activity, similar to a sub-cutaneous
injection of an antibody solution at the same dosage. Comparison
of in vitro and in vivo data suggests the antibody remains stable due
to crowding effects as it is transferred from the dispersion buffer
to similarly-crowded physiological compartments. Dispersions
of protein nanoclusters may enable patient self-administation
of biological pharmaceuticals and provide insight into protein
stability in vivo.
BIOT 385 – 2:40 p.m.
High throughput biophysical characterization of antibody
formulations
Vladimir Razinkov, [email protected], Feng He, Christopher I
Woods, Linda O Narhi, Gerald W Becker.Department of Process & Product
Development, Amgen, Seattle, Washington 98119, United States
Characterization of a formulated monoclonal antibody requires
a rigorous assessment of the molecule’s stability. Biophysical
properties often determine not only molecular attributes and
structural stability but also the connection to efficacy of the final
drug product. During the early stages of drug development it is very
important to mitigate any adverse effects that could result from the
manufacturing process, storage, transportation and administration
to patients. Here we present several applications of high throughput
methods for biophysical characterization during early formulation
development. Biophysical indicators of conformational and
colloidal stabilities were correlated with aggregation propensity
under different stresses during both short and long term stability
studies. A high throughput method for viscosity measurement
is also described as a significant part of formulation screening.
Integration of design of experiment methodology, high throughput
assays, and statistical analysis allowed us to determine the design
space for optimal formulation conditions using a minimal amount
of material and time.
BIOT 386 – 3:00 p.m.
Opalescence behaviors of a monoclonal antibody at high
protein concentrations as related to liquid-liquid phase
separation
Jifeng Zhang, [email protected] of Analytical Sciences,
Amgen, Thousand Oaks, CA 91320, United States
Recombinant therapeutic antibody solutions at high protein
concentrations often appear to be opalescent visually. Although
this phenomenon is well-known, its mechanism remains to be
elucidated. Opalescence behaviors for an IgG2 in salt solutions
have been examined at different conditions including pH, salt
concentration, temperature, and protein concentration. The
experimental results suggest that the opalescence behaviors are
dominated by critical opalescence, the enhanced density fluctuation
occurring near the vicinity of the critical point on the liquid–liquid
coexistence curve in the temperature–protein concentration phase
diagram. The enhanced density fluctuation has been confirmed
by the observation of the droplet formation using microscopy.
Away from the critical point, the opalescence behavior is more
closely related to the antibody self-association (agglomeration)
caused by the attractive interactions between protein molecules.
Furthermore, there is a surprising nonmonotonic relationship
between opalescence and salt concentration when the solution pH
is away from the pI.
BIOT 387 – 3:40 p.m.
Protein interactions, stability and phase behavior – analogies
to colloids revisited
Peter Schurtenberger, [email protected].
Department of Physical Chemistry, Lund University, SE-221 00, Lund, N/A,
Sweden
The understanding of protein solutions, their dominant interactions
and their complex phase behavior is an important topic that has
greatly profited from comparisons with colloid model systems.
Initially, these developments were primarily driven by attempts to
better understand and improve protein crystallization. However,
145
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of chromatographic steps, improve process robustness and get a
100% improvement in overall yield. High Throughput Process
Development (HTPD) with a Tecan automated liquid handling
system was used in conjunction with high throughput analytical
tools. These techniques were applied to rapidly screen binding
conditions, capacities and resolution on various resin modalities
in 96-well filter plates. Once the optimal operating windows were
determined, the resins and processing conditions were scaled
up into RoboColumns, run eight at a time on the Tecan, for
optimization of each unit operation. The optimized process was
then further scaled up to allow a comparability assessment with
the existing product.
BIOT 388 – 4:20 p.m.
High-throughput viscosity measurements using microrheology
and microfluidics
Eric M Furst, [email protected] of Chemical Engineering,
University of Delaware, Newark, DE 19716, United States
I will describe recent work on high-throughput viscosity
measurements using a combination of microrheology and
microfluidics. A series of microrheology samples is generated
as droplets in an immiscible spacer fluid using a microfluidic
T-junction. The compositions of the sample droplets are
continuously varied over a wide range. Viscosity measurements are
made in each droplet using multiple particle tracking microrheology.
I will review the key design and operating parameters, including
the droplet size, flow rates, rapid fabrication methods and passive
microrheology techniques. Validation experiments are performed
by measuring the solution viscosity of glycerine and the biopolymer
heparin as a function of concentration. Overall, the combination
of microrheology with microfluidics maximizes the number of
viscosity measurements while simultaneously minimizing the
sample preparation time and amount of material, and should be
particularly suited to the characterization of protein solution
viscosity for therapeutic agents.
BIOT 389 – 4:40 p.m.
Small-angle neutron scattering and neutron spin echo
characterization of monoclonal antibody self-associations at
high concentrations
Eric J. Yearley1, [email protected], Thomas M. Scherer3, Steve J.
Shire3, Isidro (Dan) Zarraga3, Yatin R. Gokarn3, Norman J. Wagner1, Yun
Liu2. (1) Department of Chemical Engineering, University of Delaware,
Newark, De 19716, United States (2) NIST Center for Neutron Research,
National Institute for Standards and Technology, Gaithersburg, MD 20899,
United States (3) Genentech, Inc., South San Francisco, CA 94080, United
States
Concentrated therapeutic protein formulations offer numerous
delivery and stability challenges. In response, novel Small-Angle
Neutron Scattering (SANS) and Neutron Spin Echo (NSE)
investigations have been performed to probe the protein-protein
interactions and diffusive properties of highly concentrated MAbs.
The SANS data demonstrate that the inter-particle interactions for
a highly viscous MAb at high concentrations (MAb1) are highly
attractive, anisotropic and change significantly with concentration
while the viscosity and interactions do not differ considerably for a
lower viscosity MAb (MAb2). The NSE results furthermore indicate
that MAb1 and MAb2 have strong concentration dependencies of
dynamics at high Q that are correlated to the translational motion
of the proteins. It has also been revealed that the individual MAb1
proteins form small clusters at high concentrations in contrast to
the MAb2 proteins, which are well-dispersed. Notably, MAb1 still
forms clusters even at relatively low concentrations, indicative of a
strong attraction between the proteins. contaminants and suitable
for downstream applications, such as sequencing. formation. This
poster presents data around the integrated upstream and early
purification efforts that led to an effective control of acidic variant
formation. Acceptable variant levels in the final drug substance
were achieved without any effect on process yield.
BIOT 390
Preparation of dextran modified magnetic nanoparticle and its
application in DNA detection
Li Zhiyang1,2, He Nongyue1, [email protected], Wang Fang1, Liu Bin1,
Shi Zhiyang3, Wang Hua3, Li Song1, Liu Hongna1. (1) State Key Laboratory
of Bioelectronics, Southeast University, Nanjing, Jiangsu 210096, China (2)
College of Life Science, Yangtze University, Jingzhou, Hubei 434025, China
(3) Department of Microbiology, Jiangsu Province Center for Disease Control
and Prevention, Nanjing, Jiangsu 210096, China
For reducing the steric hindrance and nonspecific binding of the
target DNA, The dextran was used as an arm to immobilize on
the surface of magnetic nanoparticles (MNPs). It was showed that
magnetic separation was the best method in preparation of dextran-
146
MNPs (DMNPs). Aspartic acid and aminated DNA probe were
modified to the dextran immobilized on the surface of DMNPs one
after another. These probe-DMNPs were applied to detect biotinlabeled PCR product of Ecoli. O157:H7 genome by hybridization.
Then these complexes were bonded with streptavidin-modified
alkaline phosphatase (ALP-SA). Finally the chemiluminescent
signals were detected by adding 3-(2-spiroadamantane)-4methoxy-4- (3-phosphoryloxy) phenyl-1, 2-dioxetane (AMPPD).
The results showed that this method had a good specificity, and
more sensitive than that of using MNPs as a solidoid carrier.
BIOT 391
Recovery of small diagnostic DNA fragments from serum using
compaction precipitation
Binh V Vu1, [email protected], Kim Anthony2, Ulrich Strych2,
Richard C Willson1,2. (1) Chemical and Biomolecular Engineering,
University of Houston, Houston, TX 77204, United States (2) Biology and
Biochemistry, University of Houston, Houston, TX 77204, United States
While most nucleic acids are intracellular, trace amounts of
DNA and RNA are present in peripheral blood. Many studies
have suggested the potential utility of these circulating nucleic
acids in prenatal diagnosis, early cancer detection, and infectious
diseases diagnosis. However, DNA circulating in blood is usually
present at very low concentrations (ng/ml), and is in the form of
small fragments (<1,000 bp), making its isolation for diagnostic
purposes challenging. Here we report an improved method for
isolation of small DNA fragments from serum using the groovebinding, polycationic compaction agents to selectively precipitate
DNA. A 151 bp Lambda DNA fragment served as the model DNA
in our experiments. Using compaction precipitation, we were able
to recover and detect very low levels (0.01 ng/ml) of a small DNA
fragment in serum. The isolated DNA product after compaction
precipitation was largely free of serum contaminants and suitable
for downstream applications, such as sequencing.
BIOT 392
Interactions of osteosarcoma cells with engineered
nanocomposite scaffolds
Jonathan Sanchez2, [email protected], Yoshira M Ayala2,
Bryan Rios2, Carmen J Hernandez2, Ipsita A Banerjee1, Nako Nakatsuka1.
(1) Chemistry, Fordham University, Bronx, New York 10458, United States
(2) Biology, University of Puerto Rico, Humacao, Puerto Rico
nanocrystals with osteosarcoma cells. In order to mimic the extracellular matrix, those materials were functionalized with collagen
and peptide sequences such as GRGDSP or BMP. The morphologies
of the formed nanocomposites were examined by various electron
microscopic methods. In some cases, the materials were bound
to self-assembled peptide nanotubes. We also attached ellagic
acid to the materials to explore its anti-carcinogenic potency on
osteosarcoma cells. In addition to conducting cytotoxicity analysis,
we also examined the immunological response of the materials in
the presence of macrophages. The cytotoxicity analysis revealed that
the ellagic acid functionalized conjugates were cytotoxic toward
osteosarcoma and its effect was concentration dependent. We
further examined the effect of the nanomaterials on the expression
of EGFR in osteosarcoma and their implication in pathogenesis.
BIOT 393
Probing the mechanism of LipL: A non-heme, mononucler,
Fe(II) dependent α-ketoglutarate:UMP dioxygenase that
initiates the biosynthesis of high carbon nucleoside antibiotics
Anwesha Goswami, [email protected], Steven Van Lanen.Department
of Pharmaceutical Sciences, University of Kentucky, Lexington, KY 405083270, United States
Several nucleoside natural product antibiotics from Streptomyces
sp. and actinomycetes have been discovered in recent years that
target bacterial peptidoglycan cell wall biosynthesis by inhibiting
the bacterial translocase I (MraY). The biosynthetic gene clusters
for A-90289, liposidomycins and caprazamycins have recently been
identified and shown to encode a protein with sequence similarity
to proteins annotated as α-KG:taurine dioxygenases (TauD). This
enzyme (LipL) has been characterized as a mononuclear, nonheme, Fe(II) dependent α-KG:UMP dioxygenase that carries out
the net dephosphorylation and two electron oxidation of prime
substrate uridine monophosphate (UMP) to uridine-5’-aldehyde
through a postulated radical mechanism involving the formation
of an unstable hydroxylated intermediate. This discussion will
focus on the synthesis and characterization of a modified UMP
with a 5’ C-P bond replacing the bridging phosphoester oxygen
yielding a poor leaving group and thereby a potential stable
hydroxylated intermediate when allowed to react with LipL. The
development of 18O2, H217O and elemental sulfur labeled UMP
is also discussed as an alternate strategy to tracking the fate of the
eliminated phosphate.
In this work, we examined the interactions of various
nanoconstructs
containing
beta-tricalcium
phosphate
nanocrystals, hydroxyapatite as well as alpha-tricalcium phosphate
147
Wednesday Afternoon
issues of interparticle interactions, aggregation, cluster and
dynamical arrest in protein solutions have to be seen in a broader
context, since they are of central importance to a variety of topics
ranging from cluster formation in various diseases to the production
of novel food systems. Understanding interparticle interactions
in protein solutions is for example of central importance to gain
insight into the origin of protein condensation diseases such as
Creutzfeldt Jakob, Alzheimer, Parkinson or cataract.
In my presentation I will illustrate how we can use light, smallangle neutron and X-ray scattering and rheology experiments
combined with molecular dynamics computer simulations to
identify, measure and model the molecular interactions and
emergent optical and viscoelastic properties and the phase
behavior of concentrated solutions of individual proteins as well
as the relevant, complex cytoplasmic mixtures. I will demonstrate
the benefits and limits of a course-grained view of proteins, where
proteins are modeled as colloid-like particles interacting via a
centrosymmetric effective interaction potential, and show how we
can incorporate a molecular viewpoint that goes beyond the simple
colloidal sphere model.
enzymes were purified and characterized.
Characterization of a G-quadruplex structure within CHRNA4
mRNA
BIOT 396
Camille A. Pottinger, [email protected], Mihaela-Rita
Mihailescu.Department of Chemistry and Biochemistry, Duquesne
University, Pittsburgh, PA 15219, United States
RNA sequences containing G-repeats can fold into G quadruplex
structures composed of stacked G tetrads. Such structures are
very stable due to the Hoogsteen-hydrogen bonding between the
guanines within each tetrad and to the presence of potassium
ions which coordinates them. Cholinergic Receptor Nicotinic
Alpha 4 (CHRNA4) messenger RNA (mRNA) encodes for the
α4 subunit of the larger protein, neuronal nicotinic acetylcholine
receptor (nAChR), found in nerve cells. The mutated CHRNA4
results in disorders such as autosomal dominant nocturnal frontal
lobe epilepsy (ADNFLE) and nicotine dependence. Based on its
sequence, which is rich in guanine nucleotides, it is possible that
the CHRNA4 messenger RNA will adopt a G quadruplex structure.
In this study we used different biophysical methods to determine
that indeed this mRNA adopts a G quadruplex structure, and to
characterize it.
BIOT 395
Activity and stability of halophilic hydrolases in nonaqueous
media
Ezio Fasoli1, [email protected], Hildamarie Caceres1, Karla
Esqulin1, Brian Rosseló1, Aramis Villafane1, Lilliam Casillas2. (1)
Department of Chemistry, University of Puerto Rico at Humacao,
Humacao, Humacao, PR 00791, Puerto Rico (2) Department of Biology,
University of Puerto Rico at Humacao, Humacao, Humacao, PR 00791,
Puerto Rico
Biocatalysis requires the use of stable and versatile enzymes that can
be exposed to a vast range of reaction conditions often deleterious
for optimal enzyme activity. Hydrolases are synthetic useful
biocatalysts because of their high operational stability, chemo- and
stereo-selectivity. In this project four metagenomic libraries and 150
microorganisms isolated from tropical solar salterns at Cabo Rojo,
Puerto Rico were screened for the presence of hydrolases (proteases
lipases, esterases, and epoxy hydrolases). Microorganisms were
cultivated in marine broth while metagenomic in LB medium
containing 4.5% NaCl. The positive microorganisms were identified
by colorimetric estimation of the released para-nitro phenol,
para-nitro aniline or adrenaline assay, using a microplate reader.
Positives were tested for different hydrolase activities in organic
solvents and ionic liquids and stability over a period of 24 hours
in 15%, 30% and 50% (v/v) concentrations. The most promising
148
Diblock copolymer foam surface chemistry influences
mesenchymal stem cell fate
Somyot Chirasatitsin1, [email protected], Priya Vishwanathan2,3,
Gwendolen Reilly3, Giuseppe Battaglia2, Adam Jeffrey Engler1. (1)
Department of Bioengineering, University of California, San Diego, La
Jolla, CA 92093, United States (2) Department of Biomedical Science, The
University of Sheffield, Sheffield S10 2TN, United Kingdom (3) The Kroto
Research Institute, The University of Sheffield, Sheffield S3 7HQ, United
Kingdom
Adhesions play an important role in cell behavior, including
differentiation. Substrates are typically modified with homogeneous
protein coatings; extracellular matrices in vivo provide
heterogeneous adhesive sites. To mimic adhesive heterogeneity,
internal phase emulsion foams were polymerized with polystyrenepolyacrylic acid (PAA) and polystyrene-polyethylene oxide
(PEO) to determine if interface de-mixing would form patchlike surfaces. PEO/PAA mole ratios were confirmed by XPS and
water contact angle while spatial distribution was measured by
chemical force spectroscopy. This method confirmed the presence
of patch-like PAA domains. Protein differentially adsorbs on PEO
and PAA, so adsorption on foam mixtures was copolymer ratio
dependent. Bone marrow-derived mesenchymal stem cell (BMSC)
adhesion was ratio dependent, but the highest density and vinculin
expression was observed for 75PEO/25PAA. BMSCs appeared to
change lineage expression the most on this composition, suggesting
that this foam, which exhibits small adhesive PAA domains, may
be more biomemetic than uniformally adhesive scaffolds, e.g.
0PEO/100PAA.
BIOT 397
Dynamics and interactions of VEGF-DNA aptamers: Ensemble
and single molecule studies
Mohan Poongavanam2, [email protected], K.
Kourentzi1, A. Potty1, I. Kanakaraj2, N. Taylor4, H. Kim3, N. Poddar4,
X. Zhang2, U. Strych2, C. Landes4, R. C. Willson1,2. (1) Department of
Chemical and Biomolecular Engineering, University of Houston, Houston,
TX 77004, United States (2) Department of Biology and Biochemistry,
University of Houston, Houston, TX 77004, United States (3) Department
of Biomedical Engineering, University of Houston, Houston, TX 77004,
United States (4) Department of Chemistry, Rice University, Houston, TX
77005, United States
We present a comprehensive investigation of the molecular
interactions between DNA aptamers aV and aH and their target,
vascular endothelial growth factor (VEGF165). Fluorescence
anisotropy, isothermal titration calorimetry and sedimentation
studies with aV aptamer indicate that, at equilibrium, a single
aptamer binds to each VEGF homodimer, suggesting an
involvement of the subunit interface in aptamer recognition.
We further present kinetics of association and dissociation for
VEGF with various aptamer mutant forms using surface plasmon
resonance. Using single-molecule intramolecular fluorescence
resonance energy transfer, we investigate the Mg2+-dependent
conformational dynamics of aV-VEGF. Without its protein target,
aV aptamer appears to favor the closed conformation, shifting
to a more open conformation upon interaction with VEGF. The
association of the aH aptamer to VEGF, by contrast, is not sequence
specific and is mediated by electrostatic interactions, sensitive to
ionic strength, and entropically-driven, compared to enthalpicallydriven aV-VEGF interaction.
BIOT 398 - Withdrawn
BIOT 399
Para-aminobenzamidine linked affinity membranes for
plasminogen activator purification
Yiaslin Ruiz1, [email protected], Nelson Rivera2, Ezio Fasoli2, ezio.
[email protected], Vibha Bansal1, [email protected]. (1) Department
of Chemistry, University of Puerto Rico at Cayey, Cayey, PR 00736, Puerto
Rico (2) Department of Chemistry, University of Puerto Rico at Humacao,
Humacao, PR 00792, Puerto Rico
Membrane based separations are emerging as a superior alternative
to packed bed chromatographic processes by virtue of high surface
area, reduced diffusion distance, low operating pressure, and higher
cost effectiveness. Their application can be particularly interesting
for large scale isolation of therapeutic proteins if the membranes
are made more selective for the protein of interest. In this paper,
an affinity membrane was designed and prepared for isolating
plasminogen activators (PAs) from mono- and multi- component
systems. PAs are therapeutic agents for thrombovascular disorders.
Regenerated cellulose (RC) membranes were chemically modified
with para-aminobenzamidine (pABA) via spacer arms of different
lengths. The membrane containing pABA linked through 14-atoms
spacer arm was found to bind up to 5.5-times higher amounts of
PA as compared to membranes linked to pABA through shorter
(5-, and 7- atoms) spacer arms, from pure PA solution as well as the
conditioned cell culture media. A 40-fold purification was achieved
in a single step separation of PA from HEK-293 conditioned media
using these affinity membranes, irrespective of the length of the
spacer arm. An extensive regeneration procedure allowed the
preservation of approximately 90% of the PA binding capacity of
the membranes even after five cycles of use.
BIOT 400
Enhanced binding on clustered-charge adsorbents: Ensemble
and single-molecule studies
Sagar P Dhamane1, [email protected], Wen-Hsiang Chen2,
Charlisa R Daniels3, Lydia Kisley3, Mohan-Vivekanandan Poongavanam1,
Katerina Kourentzi2, Christy Landes3, Richard C Willson1,2. (1)
Department of Biology and Biochemistry, University of Houston, Houston,
TX 77004, United States (2) Department of Chemical and Biomolecular
Engineering, University of Houston, Houston, TX 77004, United States
(3) Department of Chemistry, Rice University, Houston, TX 77005-1827,
United States
Traditionally, charged ligands are introduced into ion-exchange
matrices by random chemical derivitization, producing a
heterogeneous charge distribution. We demonstrated the improved
protein-binding capacity and selectivity of ion-exchange adsorbents
that display an engineered “clustered” rather than random,
distribution of surface charges. We also found that clustered-charge
(penta-argininamide and penta-lysinamide) adsorbents selectively
favor biomolecules with inherent charge clustering, such as apo-αLactalbumin, cytochrome b5 and nucleic acids. In order to prepare
cost effective clustered-charge adsorbents, we also have explored
the possibility of use of spermine, a natural polycation, as a ligand.
At the single-molecule level, we were also able to observe the
specific protein ion-exchange adsorption by direct confocal laser
scanning imaging and to quantitatively distinguish free diffusion
from hindered diffusion caused by interactions with the adsorbent
(clustered-charge anion exchanger on agarose support) using
fluorescence correlation spectroscopy.
BIOT 401
Disposable fluid management solutions for downstream
bioprocessing operations
Jakob Liderfelt1, [email protected], Annika Morrison1, William
Larsen2. (1) RTP&F, GE Healthcare, Uppsala, Sweden (2) RTP&F, GE
Healthcare, Westborough, MA 123, United States
Interest in disposables and single-use bioprocessing equipment has
increased over recent years. Today, new build pilot and production
facilities are often based on a flexible platform where unit operations
occupy only a small footprint. To increase flexibility and minimize
space required, ready-to-use plastic bags and flexible plastic tubing
increasingly replace fixed stainless steel buffer tanks and piping.
This work describes ready–to-use solutions for handling buffers
and product streams in process setups including chromatography
systems and cross flow filtration systems.
149
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BIOT 394
Micromachined multielectrode microprobes for glutamate and
dopamine with an on-probe iridium oxide reference
Vanessa M Tolosa1, Tina Tseng1, Kate M Wassum2, Allison Yorita1,
[email protected], Nigel T Maidment2, Harold G Monbouquette1,
[email protected]. (1) Chemical and Biomolecular Engineering, UCLA,
Los Angeles, CA 90095, United States (2) Psychiatry and Biobehavioral
Sciences, UCLA, Los Angeles, CA 90095, United States
We are designing, producing, and implementing micromachined,
multielectrode microprobes for the monitoring of neurotransmitter
concentration changes in near real-time in the brains of live
rodents. We construct 150 microprobe devices simultaneously
on four-inch silicon wafers with four microelectrodes per probe.
One or more microelectrode sites are chemically modified with
permselective polymer films and immobilized glutamate oxidase
for the electroenzymatic detection of glutamate. Stand-alone
glutamate biosensors also can be manufactured by a similar
process using 50-μm diameter platinum wire. However, the larger
(~150 μm) multielectrode probes permit the incorporation of
multiple functions on the same device. For example, in addition to
glutamate sensing sites, other sites may be modified for sensing of
dopamine. Alternatively, the electrodeposition of an iridium oxide
film on a site provides an on-probe reference. Such an on-probe
reference eliminates the need for a separate reference electrode and
results in a 60% decrease in signal noise.
BIOT 403
Intein-mediated bioactive protein hydrogel as scaffold for
enzyme immobilization
Miguel Ramirez, [email protected], Dongli Guan, Zhilei
Chen.Artie McFerrin Department of Chemical Engineering, Texas A&M
University, College Station, TX 77843, United States
Hydrogels made entirely of proteins/peptides or hybrid proteins/
polymers have found applications as tissue engineering scaffolds,
drug delivery carriers, controlled release depots and as key
components of biosensing and bioanalytical devices. Hydrogels
formed through a self-assembly process have the potential to
produce precisely defined, hierarchical 3D structures – a particularly
desirable trait when assembling enzymes/catalysts for cascade
reactions. Taking advantage of intein-mediated protein ligation,
we have constructed a protein hydrogel with the potential to selfassemble in situ for therapeutic applications. The building blocks
of our protein hydrogels are two protein triblock copolymers that
are expressed separately in E.coli. Each copolymer contains one
half of a naturally split intein and a crosslinker comprising trimeric
protein subunit. Upon mixing of these two protein copolymers,
150
the split intein in each copolymer covalently links the crosslinker/
bioactive protein from the two triblock copolymers, resulting in the
formation of a protein polymer molecule capable of self assembling
into a hydrogel. By choosing a highly stable trimeric protein as
the crosslinker and a highly active intein, we obtained a protein
hydrogel that is highly stable in solution. We then incorporated
a docking station peptide in the hydrogel backbone to facilitate
specific immobilization of target protein fused to a corresponding
docking protein. Target protein can be loaded into the hydrogel
either prior to or post hydrogel formation. This technology is
versatile and efficient and should be useful for many applications
including scaffold for enzyme immobilization in biofuel cells and
scaffold for cell attachment/growth in tissue engineering.
BIOT 404
HiScreen™ prepacked columns designed for process
development
Anna Heijbel, [email protected], Maria Björner, Annika Forss.GE
operations used in the manufacture of therapeutic monoclonal
antibodies (mAbs), and has been shown to provide moderate
clearance of retroviruses such as XMuLV. Protein A chromatography
can remove up to 4 LRVs (log reduction values) of retrovirus but
sometimes removes as little as 1 LRV despite the fact that virus
is not expected to bind to this resin. Additionally, LRVs obtained
by the mechanism of removal on protein A are reproducible for
a given mAb, but can vary widely for different mAbs or different
processing conditions. Little is known about how the virus
interacts with the Protein A column, mAb product or impurities
during the operation of the step. Experiments were performed to
study the behavior of XMuLV on a MabSure Protein A column.
The results show that virus alone has little to no interaction with
the column, but in the presence of HCCF (harvested cell culture
fluid), a portion of the virus co-elutes with the product. Further
studies to determine the impact of mAb, impurities, and column
washes will be presented.
BIOT 406
Healthcare Bio-Sciences AB, Uppsala, Sweden
Scalable manufacturing processes for therapeutic stem cells
Development of purification processes is done in small scale due
to sample consumption, convenience, time and cost. The aim is
to obtain an efficient, robust and scalable process with highest
possible throughput at lowest cost. HiScreen prepacked columns
are available with >30 different BioProcess™ media. Column
volume is 4.7 ml, bed height 10 cm, making them suitable for
process development. Two columns can easily be connected in
series giving a bed height of 20 cm. All media prepacked in the
HiScreen columns are available in other different prepacked
formats and bulk packs, making it possible to use the same medium
for development work, pilot studies and routine production.
Here is presented media screening of nine different HIC media and
four different anion exchangers (Q ligand). Also 40 times scaling
up from HiScreen via HiScale™ to AxiChrom™ column is presented
at two different residence times. The results show robustness and
reproducibility.
Lye Theng Lock, [email protected], Jacob Pattasseril, Elizabeth
Misleh, Jon Rowley.Cell Processing Technology, Lonza Walkersville,
Walkersville, Maryland 21793, United States
BIOT 405
Understanding XMuLV clearance on Protein A
chromatography
Julia Bach1, [email protected], Beth Larimore1, Shivanthi Chinniah2,
Lisa Connell-Crowley1. (1) Department of Purification Process
Development, Amgen, Seattle, Washington 98119, United States (2)
Department of Cellular Resources, Amgen, Seattle, Washington 98119,
United States
Protein A affinity chromatography is one of the most common unit
Therapeutic cell manufacturing differs from traditional biologicbased drugs in that the final product is a living, biologically
functional cell. As primary and stem cell-based therapeutics are
progressing through clinical trials, there are three main challenges
facing this new class of products. These challenges include scaling
up traditional small scale culture processes while maintaining
the phenotype and biological functionalities of the cell products,
establishing technologies for downstream processing to address
new process bottlenecks, and controlling the Cost of Goods
(CoGs) of these inherently expensive products. Here, we have
characterized hMSC production from 10- to 40-layer platforms
and demonstrated comparable level of flow markers (CD105,
CD166 and CD45) and secreted cytokine (IL-6, IL-8 and VEGF
in pg/cell/day). Furthermore, tangential flow filtration (TFF) was
developed as a closed, scalable cell harvest and concentration
system, with shear rate being the main critical process parameter
controlling final cell viability, and must be maintained <4000 sec-1.
BIOT 407
Parallel synthesis and screening of endotoxin-binding
polymers
Thrimoorthy Potta, [email protected], Divya Nair, Gabriela
Montanez, Kaushal Rege.Chemical Engineering, Arizona State University,
TEMPE, AZ 85281, United States
Endotoxins are complex polysaccharide molecules present on
outer membranes of gram-negative bacteria. Endotoxins play a
vital role in the pathogenesis of septic shock, which is a common
clinical problem and leading cause of mortality in immunologically
susceptible patients. In addition, these anionic biomacromolecules
are common contaminants in E.coli culture broths used for the
generation of biological products. In this study, a library of cationic
polymers was generated using ring opening polymerization
between various amines and diepoxides. Synthesized polymers
were purified, characterized using various analytical techniques
including 1H-NMR and FTIR, and screened for their endotoxin
binding abilities at different salt concentrations. A fluorescencebased screen led to identification of lead polymers which can
successfully bind endotoxin. Polymer-endotoxin complexes were
characterized by particle size and zeta potential analyses, and
polymer-endotoxin binding was verified using additional methods.
These results suggest that polymers developed in this study can be
employed for the sequestration of endotoxins from culture broths
in biological separations.
BIOT 408
Diblock copolymers with tunable pH transitions for gene
delivery
Matthew J Manganiello, [email protected], Connie Cheng,
Anthony J Convertine, James D Bryers, Patrick S Stayton.Department of
Bioengineering, University of Washington, Seattle, WA 98195, United States
A series of diblock copolymers containing an endosomal-releasing
segment composed of diethylaminoethyl methacrylate (DEAEMA)
and butyl methacrylate (BMA) at varying molar feed ratios and
a plasmid DNA-condensing segment of dimethylaminoethyl
methacrylate (DMAEMA) were synthesized via reversible additionfragmentation chain transfer (RAFT) polymerization. These
diblock copolymers self-assemble into micelles at physiological
pH but undergo a phase transition at lower pH values. The pH at
which this transition occurs can be precisely tuned by modification
of the BMA content. Diblock copolymers with 30 – 40 % BMA
content exhibited phase transitions at pH values similar to those
encountered in the early and late endosomes. These materials
showed significant levels of red blood cell lysis at these pH values
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BIOT 402
BIOT 409
Modified calcium surfaces as optimum tissue scaffolds
Jared D Romeo1, [email protected], Rachelle N Palchesko1,
Kenneth A McGowan2, Ellen S Gawalt1. (1) Department of Chemistry
and Biochemistry, Duquesne University, Pittsburgh, PA 15282, United
States (2) Westmoreland Advanced Materials, Monessen, PA 15062, United
polymer films of polymethyl methacrylate (PMMA) and four-vinyl
pyridine (P4VP) and on electrospun fibers of these materials, whose
fiber radii and spacing was varied. Addition of dexamethasone
promoted differentiation on all substrates, as expected. In the
absence of dexamethosone, spontaneous differentiation was
not observed on any of the flat films. On PMMA substrates
differentiation was observed only on fibers with diameters less than
one micron, regardless of surface treatment, while copious amounts
of hydroxyapatite (HA) were observed on all P4VP fibers. Closer
examination revealed that the HA deposition was templated along
the polymer fibers, where a secondary structure of ECM protein
fibers was also present. In order to determine the differentiation
pathways induced, odontoblast and osteoblast markers are being
evaluated by RT-PCR.
States
Providing an optimal tissue scaffold is critical for the regrowth
of bone at a major injury site. In this study, calcium aluminate
(CaAl) and hydroxyapatite, the mineral components of bone, were
investigated as potential bone tissue scaffolds. These materials are
bioactive, easily synthesized at room temperature, and have been
shown to induce de novo bone formation. To enhance regenerative
properties, the scaffolds were modified via chemical immobilization
of bone morphogenetic protein 2 (BMP2), a protein responsible
for enhancing bone cell growth and differentiation, and the cell
adhesion peptide Lys-Arg-Ser-Arg (KRSR). Viability of both
fibroblast, a component of connective tissues, and osteoblast cell
lines was examined to determine cell-binding specificity. This
study effectively shows that BMP2 functionalized and KRSR
functionalized calcium scaffolds selectively increase osteoblast
viability as compared to fibroblast viability and enhance cell
adhesive properties over unmodified scaffolds.
BIOT 410- Withdrawn
BIOT 411
Role of surface chemistry and morphology in dental pulp stem
cell differentiation
Giulia Suarato1, [email protected], A.K. Bherwani2, Chung-
Chue Chang1, Aaron Akhavan3, Marcia Simon2, Miriam Rafailovich1. (1)
Materials Science, Stony Brook University, Stony Brook, New York 11794,
United States (2) Oral Biology/School of Dental Medicine, Stony Brook
University, Stony Brook, New York 11794, United States (3) RAMBAM
Mesivta High School, Lawrence, New York, United States
The relative influence of chemistry and morphology on the
induction of dental pulp stem cells (DPDC) along osteogenic
lineage was investigated. DPSC were plated on spun cast flat
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BIOT 412
Label-free screening of multiple cell-surface antigens using
single micropores
Anand Kesavaraju1, [email protected], Matthew R Chapman2,
Karthik Balakrishnan3, Lydia L Sohn3. (1) Department of Bioengineering,
University of California, Berkeley, Berkeley, CA 94720, United States (2)
Department of Biophysics, University of California, Berkeley, Berkeley,
CA 94720, United States (3) Department of Mechanical Engineering,
University of California, Berkeley, Berkeley, CA 94720, United States
Pore-based biosensing has emerged as a versatile label-free
screening technique. Recent work has shown that functionalizing a
pore with specific antibodies can lead to slower transit time for cells
expressing the complementary surface antigen when compared
to the transit time of cells passing through a pore functionalized
with isotype control antibody. This label-free method is ideal for
screening small populations of rare cells and applications where
recovery of the cells for downstream analysis and culture is required.
However, functionalization with only one antibody at a time limits
the utility of this technique. To this end, we have re-designed an
artificial pore that is divided into three separate sections. Each
section can be functionalized with a different antibody allowing
us to perform label-free multianalyte screening of cells. We will
discuss the application of this innovative pore to screen stem cells
for multiple cell-surface antigens and the characterization of nicheto-niche heterogeneity.
BIOT 413
BIOT 414
Asymmetry-based selection algorithm for designing highly
active siRNAs
Column-free, protease-free protein purification via mini-intein
cleavage
Amanda P. Malefyt1, [email protected], Ming Wu2, James SimsWright5, Arul Jayaraman4, Kyongbum Lee5, Christina Chan1,3, S. Patrick
Walton1. (1) Department of Chemical Engineering and Materials Science,
Michigan State University, East Lansing, Michigan 48824, United States
(2) Department of Computer Science and Engineering, Michigan State
University, United States (3) Department of Biochemistry and Molecular
Biology, Michigan State University, United States (4) Department
of Chemical Engineering, Texas A&M University, United States (5)
Department of Chemical and Biological Engineering, Tufts University,
Miguel Ramirez, Najla Valdes, Zhilei Chen, [email protected]
McFerrin Department of Chemical Engineering, Texas A&M University,
United States
In the development of RNA interference (RNAi) therapeutics,
selecting siRNA sequences that complement the messenger RNA
(mRNA) target do not guarantee silencing. Factors such as 5’ end
stability are known to be critical for ensuring the correct strand
is preferentially incorporated into the RNA induced silencing
complex (RISC). Two methods for determining this asymmetry
between strands are terminal sequence and relative terminal
thermodynamic stability. Through the analysis of large siRNA
databases, we have shown that highly active siRNA sequences
are more likely to have large asymmetry between the sense and
antisense 5’ ends in both end sequence nucleotides as well as
thermodynamic stability.
We used this information to create an algorithm for predicting
highly active siRNA sequences against desired proteins using
only the mRNA sequence of the target. The algorithm uses end
sequence and thermodynamic stability parameters, trained from
existing siRNA activity databases, to rank the probability that
an siRNA sequence has high, medium, and low activity for its
target gene. We will discuss the applicability of the algorithm for
predicting highly active sequences for both exogenous (Enhanced
green fluorescent protein, EGFP) and endogenous proteins (RNAdependent Protein Kinase, PKR, and Pyruvate Carboxylase, PC).
We will highlight comparisons between our technique and other
selection approaches as well as discuss methods for isolating
additional parameters to further improve algorithm accuracy.
College Station, TX 77843, United States
Current affinity tag-based protein purification approaches are not
ideal for biotechnological applications. We report the development
of a column-free, protease-free protein purification technology
that takes advantage of an engineered split-intein and a stimulusresponsive elastin-like-peptide (ELP) tag. Under reducing
condition, this engineered split-intein catalyzes trans-splicing
reaction pH 6 and a single C-terminal cleavage reaction at pH 8.
The ELP is fused to the N-terminal fragment of the split intein
and the protein of interest (POI) to the C-terminal fragment of
the intein. These two fusion proteins are expressed individually
in E. coli and mixed after cell lysis. Under low salt non-reducing
conditions, the N- and C-inteins associate strongly with each
other without cleavage, physically connecting the ELP to POI.
High salt conditions trigger ELP-mediated phase separation of the
POI. Next, the precipitate containing the POI is resuspended in a
low-salt, reducing buffer to resolubilize the aggregate and induce
intein cleavage, releasing the POI from the ELP-intein complex.
Finally, the resulting mixture is subjected to high salt conditions
again to precipitate out the undesired ELP-intein complex from
the solution, leaving only purified POI in the solution phase. This
protein purification technology is simple, convenient and rapid.
The entire process from soluble cell lysate to purified tag-free
protein takes <4h. This technology should facilitate cost-effective
purification of proteins on a large scale.
BIOT 415
Electrospun fibrous scaffolds promote breast cancer cell
alignment and epithelial-mesenchymal transition
Sharmistha Saha1, Xinrui Duan1, [email protected], Laying
Wu1, Pang-Kuo Lo2, Hexin Chen2, Qian Wang1. (1) Department of
Chemistry and Biochemistry, University of South Carolina, Columbia,
South Carolina 29208, United States (2) Department of Biological Sciences,
University of South Carolina, Columbia, South Carolina 29208, United
States
The rigidity and topography of the ECM environment have been
reported to alter cancer cell behavior. But the complexity of the in
vivo system makes it difficult to isolate and study such extracellular
topographical cues that trigger cancer cells’ response. In this work
we created polymeric electrospun fibrous scaffold with random
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but negligible cell lysis under physiological conditions. High levels
of DNA transfection were observed for the copolymer compositions
exhibiting the sharpest pH transitions and membrane destabilizing
activities, demonstrating the importance of tuning the endosomalreleasing segment composition.
BIOT 416
Electric control of enzymatic activity through redox mediators
Tanya Gordonov1, [email protected],
Eunkyoung Kim2, Gregory Payne1,2, William Bentley1,2. (1) Fischell
Department of Bioengineering, University of Maryland, College Park,
MD 20740, United States (2) Institute for Bioscience and Biotechnology
Research, University of Maryland, College Park, MD 20740, United States
As researchers seek insights into biology through an ever-shrinking
spyglass, the ability to easily control biomolecular interactions
on the nanoscale is becoming a necessity. Control over the
activity of biomolecules, such as enzymes, with redox-responsive
active sites, can be achieved by the addition of mediators with
appropriate potentials. The form of these mediators, and therefore
their reactivity with said biomolecules, can in turn be electrically
controlled. Here we present a technique where enzymatic activity
is modulated by the introduction of an electric current. The current
converts a redox mediator to a form which can change the state
of the metal center on an enzyme and therefore change its ability
to catalyze a reaction. Several redox mediators have been tested
and can be used to achieve a desired degree of control, to ensure
specificity, and to avoid unwanted effects. This advancement can
be applied to next-generation lab-on-chip platforms, where precise
control of activity or product amount is desirable. In addition, this
method can be utilized for measuring the effects of antioxidants,
studying biological signaling, and bringing bioelectronic
interconnectedness down to the nanoscale.
BIOT 417
IκBα, the most abundant inhibitor protein of the NF-κB
transcription factor, has six ankyrin repeats (ARs) in its AR
domain: four well-folded ARs (repeats 1 through 4) and two weakly
folded ARs (repeats 5 and 6). The AR 5-6 conformational flexibility
is critical for regulating NF-κB signaling. Single-molecule FRET
experiments probing the weakly folded region demonstrated
that the native state of IκBα transiently populates an intrinsically
disordered state characterized by a more extended structure and
slow millisecond-scale fluctuations. Systematic placement of
FRET fluorophores along ARs revealed a sequential unfolding of
the native state of IκBα that starts with the C-terminal AR 6 at
room temperature, followed by AR 5 at 37 °C. Fluctuations in the
intrinsically disordered AR 5-6 region correspond with the ability
of IκBα to dissociate NF-κB from DNA transcription sites, and
could provide the basis for new therapies to numerous diseases.
BIOT 418
Light-regulation of DNA editing
Chungjung Chou, [email protected], Alexander Deiters.Chemistry,
North Carolina State university, Raleigh, North Carolina 27606, United
States
Photochemical control over protein activity provides a “synthetic
switch” to manipulate cellular function with unprecedented
precision to study cell at a subcellular level. Light-activatable
proteins can be engineered in vivo by installing a photocaging
group unnatural amino acid mutagenesis. Non-damaging UV
irradiation can be used to remove the caging group and trigger
activities.
The zinc finger nuclease (ZFN) has shown great potential in gene
therapy applications. In order to accomplish spatio-temporal gene
editing, we generated a light-activated ZFN enzyme by blocking
the DNA-binding interface of the FokI nuclease module through
site-specific installation of a single caging group. Co-transfection
experiments of the caged ZFN and a suitable reporter construct
revealed that light-triggered recombination can be achieved. In
summary, we present a light-inducible ZFN that may decrease
cytotoxity and increase accuracy. The strategy may be applied to
other engineered nucleases to enable the light-induced mutagenesis
and recombination in cells and model organisms.
Nanospring dynamics and sequential unfolding of the IκBα
ankyrin repeat domain
BIOT 419
Jorge A Lamboy1, [email protected], Hajin Kim2, Taekjip Ha2,
Characterization of a monoclonal antibody product related
contaminant with three light chain subunits
Elizabeth A Komives1. (1) Department of Chemistry & Biochemistry,
University of California, San Diego, La Jolla, CA 92093, United States (2)
Department of Physics and Center for Physics in Living Cells, University of
Illinois, Urbana-Champaign, Urbana, IL 61801, United States
154
Paul Casaz, [email protected], Trevor J Morin, John Que, Greg
Babcock, Sadettin S. Ozturk, William Thomas.MassBiologics, Mattapan,
MA 02126, United States
A new product-related contaminant was discovered during the
analysis of a human monoclonal IgG1 antibody produced in
CHO cells. The novel species contains three light chains and two
heavy chains rather than two light and two heavy chains but is
distinct from the recently described “Triple Light Chain Antibody”
(Biotechnol Bioeng. 105(4 ):748-760). The structure of this species
was characterized by size exclusion chromatography, multiangle
light scattering, capillary gel electrophoresis and amino terminal
protein sequencing. Antibody binding to its target was measured
by ELISA and interferometry (Octet). The novel species eluted
between monomeric and dimeric antibody in size exclusion HPLC,
and has a mass of 176 kDa. Analysis of the novel species by reducing
and nonreducing capillary SDS gel electrophoresis demonstrated
the presence of a noncovalently bound subunit with the same
molecular weight as light chain. The stoichiometry was one
noncovalently bound subunit per antibody monomer, and three
light chains to two heavy chains. Edman degradation sequencing
confirmed that the extra subunit was human light chain. Binding
to target antigen in an ELISA was approximately 15% the level of
purified monomeric antibody. A similar difference was found in
the dissociation constants measured by interferometry. The three
light chain contaminant is present early in CHO cell culture and
copurified with monomer during protein A, anion and cation
exchange chromatography. The contaminant could be separated
from monomer by hydrophobic interaction chromatography.
BIOT 420
Adult dental pulp stem cell (DPSC) differentiation on surfaces
with mechanical patterns
Chungchueh Chang1, [email protected], Yingjie Yu1, Aneel
Bherwani2, Vladimir Jurukovski1, Betina Ferreira1, Marcia Simon2,
Zohar Bachiry4, John Jerome3, Miriam Rafailovich1. (1) Department of
Materials Sciences and Engineering, Stony Brook University, Stony Brook,
NY 11794, United States (2) Department of Oral Biology and Pathology,
Stony Brook University, Stony Brook, NY 11794, United States (3) Suffolk
County Community College, Brentwood, NY 11717, United States (4)
Yeshiva University High School for Girls, Holliswood, NY 11423, United
States
We’ve shown DPSCs can be induced to differentiate along osteogenic
pathways by altering the mechanics of substrates on which they
are cultured. In vivo, the cellular environment is heterogenous,
therefore we chose to investigate the influence of imposing a pattern
where mechanical properties are varied. Patterns were imprinted
into Si wafers by sputtering through polymer blend films spun-cast
onto Si. The residual polymer was removed by annealing at 750C. A
200nm thick Polybutadiene film was spun-cast on these patterned
surfaces. Since Tg of polybutadiene is above ambient, the polymer
flows and covers the pattern, forming areas varying in thickness.
DPSCs were incubated on patterned and flat film surfaces for 21
days. Cell morphology was determined by confocal microscopy
and was found to conform to the patterns. SEM/EDX analysis
indicated that only cells on the mechanically patterned regions
showed Hydroxyapatite deposits, indicating that just mechanical
without chemical heterogeneity can induce differentiation.
BIOT 421
Structure and function studies of heliobacterial cytochrome bc
complex and its diheme c-tpye cytochrome component
Hai Yue, [email protected], Yisheng Kang, Xinliu Gao, Hao Zhang, Robert
E Blankenship.Chemistry and Biology, Washington University in St. Louis,
Saint Louis, Missouri 63130, United States
Heliobacterium modesticaldum is a gram positive, anaerobic,
anoxygenic photoheterotrophic bacterium. Its cytochrome
bc complex (Rieske/cyt b complex) has some similarities to
cytochrome b6f complexes from cyanobacteria and chloroplasts,
and also shares some characteristics of typical bacterial cytochrome
bc1 complexes. One of the unique factors of the heliobacterial
cytochrome bc complex is the presence of a diheme cytochrome
c instead of the monoheme cytochrome f in the cytochrome b6f
complex or the monoheme cytochrome c1 in the bc1 complex. To
understand the structure and function of this diheme cytochrome
c protein, we expressed the N-terminal transmembrane-helixtruncated soluble H. modesticaldum diheme cytochrome c in
Escherichia coli. This 25 kDa recombinant protein possesses
two c-type hemes, confirmed by mass spectrometry and a
variety of biochemical techniques. Sequence analysis of the H.
modesticaldum diheme cytochrome c indicates that it may have
originated from gene duplication and subsequent gene fusion, as in
cytochrome c4 proteins. The recombinant protein exhibits a single
redox midpoint potential of + 71 mV vs NHE, which indicates that
the two hemes have very similar protein environments.
BIOT 422
Comparison of screening and response surface experimental
designs in selecting an optimized formulation for HEWL
activity
Paola M. Mendez1, [email protected], Adeola O. Grillo2. (1)
Department of Chemistry, University of the Incarnate Word, San Antonio,
TX 78209, United States (2) Department of Pharmaceutical Sciences, UIW
- Feik School of Pharmacy, San Antonio, TX 78209, United States
Factorial design of experiments (DoE) allows for the simultaneous
testing of multiple variables during screening and optimization of
processes and products. The effectiveness of different screening
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and aligned fiber orientations in order to mimic the 3D structure
of the natural extracellular matrix (ECM). Breast cancer cells were
cultured on these fibrous scaffolds for 3-5 days. The cells showed
elongated spindle-like morphology in the aligned fibers whereas
kept mostly flat stellar shape in the random fibers. Gene expression
profiling of these cells post seeding, showed up-regulation of TGFβ1, along with mesenchymal biomarkers suggesting that these cells
are undergoing epithelial-mesenchymal transitions in response
to the polymer scaffold. The results of this study indicate that the
topographical cue may play a significant role in tumor progression.
BIOT 423
Establishment of inducible iPS cells for tissue engineering for
cancer therapy
Allen L. Miao1, [email protected], Wen-hsuan Chang2, Wange
Lu2. (1) Department of Biochemistry and Molecular Biology, University
of Southern California (Summer Intern), Los Angeles, CA 90033, United
States (2) Department of Biochemistry and Molecular Biology, University
of Southern California, Los Angeles, CA 90033, United States
Tissue engineering to replace cells lost in cancer therapy is
important because surgery and radiation usually cause tissue loss
and damage. Tissue from donors can cause immune repulsion.
Converting somatic cells, such as skin fibroblast from cancer
patient to induced pluripotent stem (iPS) cells, makes it possible
to generate tissues for autologous transplantation without causing
immune response. We introduced Oct4, Sox2, and Klf4 genes,
whose expression is under doxycycline-inducible promoters, into
skin fibroblast using lentivirus to generate human iPS cells. These
iPS cells have similar characteristics with human ES cells. We also
differentiated these human iPS cells into fibroblast cells which can
be induced to iPS cells upon doxycycline treatment. The inducible
iPS cell system not only allow us to use these cells for replacement
therapy, but also provide an inducible system to investigate the
molecular mechanisms of iPS cell induction.
BIOT 424
Quality by design case studies: Integration of cell culture,
primary recovery and purification process characterization
Marlene Castro-Melchor, [email protected],
Timothy Diehl, Terrance Carone, Patrick Thompson, Susan Abu-Absi.
Manufacturing Sciences & Technology, Bristol-Myers Squibb, Syracuse, NY
13221, United States
156
The primary point of control for many critical quality attributes
is the production bioreactor. The purification steps are designed
to ensure the purity of the final drug substance through the
removal of process- and product-related impurities such as host
cell protein, host cell DNA, cell culture media components and
high molecular weight (HMW) species. Other aspects of the
protein, such as glycosylation and charge profile, are not typically
modified via chromatography steps, although there are exceptions.
The in-process hold points can also alter product quality (e.g.
charge profile) depending on the conditions during the hold,
such as pH and temperature. Each unit operation is characterized
independently using scale-down models of the bioreactor,
centrifuge and chromatography columns. Mathematical models
are generated to predict the effects of input parameters on product
quality at each step. The final control strategy, however, must
encompass the combined impact of the entire process including
raw material, bioreactor, chromatography, filtration, in-process
hold and drug substance handling effects. This paper will present
case studies from Bristol-Myers Squibb highlighting the integrated
approach we have utilized to develop control strategies for biologics
manufacturing processes.
After defining the acceptable ranges for the input/operating
parameters in the production bioreactor and primary recovery
steps, the expected variation in CQAs at harvest can be predicted
from the process characterization models. These worst-case
outputs in the harvest are evaluated for their predicted impact
to the performance of the capture chromatography step and
so on through subsequent steps of the process. Once the worstcase region of the design space is identified for a CQA, studies
are conducted at laboratory or pilot scale linking upstream and
downstream steps to demonstrate adequate control of the CQA.
Several examples of integrated design space mapping and process
improvements for better control of host cell protein, HMW and
charge profile will be presented.
BIOT 425
Recovery of small diagnostic DNA fragments from serum using
compaction precipitation
Binh V Vu1, [email protected], Kim Anthony2, Ulrich Strych2,
Richard C Willson1,2. (1) Chemical and Biomolecular Engineering,
University of Houston, Houston, TX 77204, United States (2) Biology and
Biochemistry, University of Houston, Houston, TX 77204, United States
While most nucleic acids are intracellular, trace amounts of
DNA and RNA are present in peripheral blood. Many studies
have suggested the potential utility of these circulating nucleic
acids in prenatal diagnosis, early cancer detection, and infectious
diseases diagnosis. However, DNA circulating in blood is usually
present at very low concentrations (ng/ml), and is in the form of
small fragments (<1,000 bp), making its isolation for diagnostic
purposes challenging. Here we report an improved method for
isolation of small DNA fragments from serum using the groovebinding, polycationic compaction agents to selectively precipitate
DNA. A 151 bp Lambda DNA fragment served as the model DNA
in our experiments. Using compaction precipitation, we were able
to recover and detect very low levels (0.01 ng/ml) of a small DNA
fragment in serum. The isolated DNA product after compaction
precipitation was largely free of serum contaminants and suitable
for downstream applications, such as sequencing.
BIOT 426
Prediction and performance of amorphous metal halide-based
ionic liquid pharmaceutical formulations
Katherine S. Lovejoy1, [email protected], Geraldine M. Purdy1,
Cynthia A. Corley3, John S. Wilkes3, Andrew T. Koppisch2, Rico E. Del
Sesto1. (1) Materials Physics and Applications-Materials Chemistry
Division (MPA-MC), Los Alamos National Laboratory, Los Alamos,
NM 87545, United States (2) Department of Chemistry & Biochemistry,
Northern Arizona University, Flagstaff, AZ 86011, United States (3)
Department of Chemistry, US Air Force Academy, USAF Academy, CO
80840, United States
Formulation of cationic pharmaceuticals as amorphous Lewis
acidic ionic liquids with zinc chloride is demonstrated. We predict
additional favorable interactions between other biologically
compatible metal halides and cationic drugs and biomolecules
based on principles of inorganic chemistry in aqueous solutions.
Multiple species and oligomeric forms of zinc chloride anions
depress melting and glass transition points by 100-200°C from
those of the standard chloride salts of the pharmaceutical
agents. The metal halide-based ionic liquids show indications of
improved shelf life, and should also alter bioavailability, eliminate
problems of crystal polymorphism, and allow pharmaceuticals
to be administered in liquid form for use in lotions or pediatric
medicine. The combination of two FDA-approved components
to form new ionic liquids should facilitate translation of this
strategy to commercial use. Pharmaceutical agents obtainable as
chloride salts with melting points less than about 200°C are reliably
formulated as amorphous ionic liquids using our strategy.
BIOT 427
Use of arginine and lysine as additives in final formulation to
increase process performance and product stability
Denise Tanis, [email protected], Luca Di Noto, Rachael Alford.
Purification Development, Alexion Pharmaceuticals, Cheshire, CT 06410,
United States
Ultrafiltration/Diafiltration (UF/DF) is commonly used in biotech
industry to formulate therapeutic proteins into final delivery buffer.
UF/DF can be executed using tangential flow filtration (TFF)
technology. During this process, the protein of interest interacts
with the TFF membrane generating proteinaceous gel layer on the
membrane surface that can generate a flux decay decreasing process
efficiency. This problem can become more apparent with proteins
that have high tendency to self aggregate or bind to hydrophobic
surfaces. In this study we used such a protein and tested its
formulation by UF/DF using arginine or lysine as additives in the
diafiltration buffer. The results show decreased flux decay and a
shorter processing time when arginine or lysine are present in the
diafiltration buffer. Additionally the protein formulated in arginine
and lysine show higher solubility and little to no aggregation when
compared to the protein in the same buffer without any of these
additives.
BIOT 428
Preparation of dextran modified magnetic nanoparticle and
its application in DNA detection
Li Zhiyang1,2, He Nongyue1, [email protected], Wang Fang1, Liu
Bin1, Shi Zhiyang3, Wang Hua3, Li Song1, Liu Hongna1. (1) State Key
Laboratory of Bioelectronics, Southeast University, Nanjing, Jiangsu
210096, China (2) College of Life Science, Yangtze University, Jingzhou,
Hubei 434025, China (3) Department of Microbiology, Jiangsu Province
Center for Disease Control and Prevention, Nanjing, Jiangsu 210096, China
For reducing the steric hindrance and nonspecific binding of the
target DNA, The dextran was used as an arm to immobilize on
the surface of magnetic nanoparticles (MNPs). It was showed that
magnetic separation was the best method in preparation of dextranMNPs (DMNPs). Aspartic acid and aminated DNA probe were
modified to the dextran immobilized on the surface of DMNPs one
after another. These probe-DMNPs were applied to detect biotinlabeled PCR product of Ecoli. O157:H7 genome by hybridization.
Then these complexes were bonded with streptavidin-modified
alkaline phosphatase (ALP-SA). Finally the chemiluminescent
signals were detected by adding 3-(2-spiroadamantane)-4methoxy-4- (3-phosphoryloxy) phenyl-1, 2-dioxetane (AMPPD).
The results showed that this method had a good specificity, and
more sensitive than that of using MNPs as a solidoid carrier.
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and optimization experimental designs for determining optimum
formulations for hen egg white lysozyme (HEWL) activity
were compared. The excipients tested were: pH, NaCl, sucrose,
polysorbate 80, and methionine. HEWL enzymatic activity was
analyzed via UV spectroscopy of Micrococcus lysodeikticus cell
lysis in plate reader format. The screening experimental designs
tested: 2-level full factorial, fractional factorial, and PlackettBurman designs, gave poor fits to the data, most likely due to
curvature in the responses. Central Composite and Box-Behnken
response surface designs of the five factors were then compared.
Both experimental designs identified significant curvature in pH
and NaCl responses. There were some differences in significant
factors and interactions identified by the two designs. Finally,
optimized formulations from the two designs will be compared.
Effects of basic amino acids on mixed-mode chromatography
Abraham Friedman, [email protected], Luca Di Noto, Rachael
Alford.Purification Development, Alexion Pharmaceuticals, Cheshire, CT
06410, United States
Mixed-mode chromatography is often used as a capture step
during downstream purification. We use a mixed mode resin
functionalized with ligands exhibiting both electrostatic and
hydrophobic properties to capture a recombinant protein that
exhibits some hydrophobic properties. Optimal desorption of this
protein from the resin requires pluripotent buffers able to modulate
two or more interactions between solute and solid phase. This study
explores the use of basic amino acids in elution buffers at different
conductivities to exploit their potential to disrupt hydrophobic
interactions. The results show an increased rate of desorption
resulting in more concentrated elution pools and reduced elution
volumes thus increasing process efficiency in this purification step.
Furthermore, the elution in the presence of these additives does
not affect the clearance of host cell impurities when compared to
elution driven only by changes in conductivity.
BIOT 430
Screening fibroblast growth factor: Fibrobast growth
factor receptor interactions with variable heparin-like
polysaccharides
Eric Sterner1, [email protected], Luciana Meli1, Jonathan S. Dordick1,2,
Robert J. Linhardt1,2,3. (1) Department of Chemical and Biological
Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180,
United States (2) Department of Chemistry and Chemical Biology,
Rensselaer Polytechnic Institute, Troy, New York 12180, United States (3)
Department of Biology, Rensselaer Polytechnic Institute, Troy, New York
12180, United States
The ability of Fibroblast Growth Factor (FGF) to bind and
dimerize with a receptor (FGFR) is mediated by heparin, a linear
polysaccharide with primarily N-sulfated and 2-O and 6-O
sulfated domains. To better understand the relationship between
FGF:FGFR:polysaccharide interactions, we have developed a highthroughput platform that can be used to simultaneously screen a
wide variety of variably sulfated polysaccharides for their ability
to promote cellular activities. Highly sulfated polysaccharides
(heparin and heparan sulfate) were compared against moderately
sulfated polysaccharides (N-sulfo-heparosan), fully desulfated
polysaccharides (N-acetyl-hepoarsan and an unmodified
decasaccharide), and truncated ultra-low molecular weight
heparins. Polysaccharides of varying length were prepared with sitespecific N-, 2-O, and 6-O sulfations and screened for their biological
158
activities. Additionally, low-molecular weight compounds were
screened for their ability to interrupt FGF:FGFR:heparin complex
formation and prevent cellular proliferation. The aforementioned
polysaccharides were screened against multiple FGF-FGFR
combinations and their effectiveness assessed via resulting cellular
proliferation.
BIOT 431
Development of a new grafting technology for the production
of cation exchange materials for downstream processing of
monoclonal antibodies
Jesper Hansson, [email protected], GE Healthcare, Life
Sciences, Uppsala, Sweden
The downstream processing of monoclonal antibodies presents
several challenges. One of the major goals of the purification
process is to reduce the amount of aggregates. Typically cation
exchange chromatography is included in the purification scheme
to address this problem.
This poster will give an overview of a new grafting technology
developed at GE Healthcare that enables attachment of polymeric
surface extenders with cation exchange functionality onto agarose
beads for use in downstream processing of monoclonal antibodies.
How the design of the surface extenders affects the performance
in terms of aggregate removal and dynamic binding capacity
will be discussed. Furthermore, benchmarking results where
the performance of existing products will be compared to our
prototypes will also be given.
BIOT 432
Influence of slight variations of ion exchange media on the
separation of proteins
Michael S Schmidt1, Lothar Jacob1, [email protected],
Heiner Graalfs1, [email protected], Christian Frech2. (1)
Department of Research and Development: Chromatography Media,
Merck Millipore, Darmstadt, Germany 64293, Germany (2) Department
of Biochemistry, Mannheim University of Applied Sciences, Mannheim,
Germany 68163, Germany
Ion-exchange chromatography (IEC) is a very versatile protein
separation method, and widely used as production scale protein
purification process. When IEC is used for high resolution
separation of proteins, retention and peak broadening are not
always reproducible and are very sensitive to small changes in
typical operating factors such as pH, ionic strength and stationary
phase ion-exchange capacity.
As lot to lot variations of ion-exchange capacity (Λ) are
unavoidable the influence of the variation of Λ was investigated.
Isocratic as well as gradient elution data of monoclonal antibodies
at different pH values on two strong anion exchange materials
(Fractogel TMAE, Fractogel TMAE HiCap) are used to evaluate the
influence of ionic capacity on protein retention. Retention models
for ion exchange chromatography based on the work of Mollerup
and Yamamoto are used to calculate their model parameters and
to predict the influence on critical process parameter like ionic
strength of elution, elution volume and purity of the final product.
BIOT 433
Overloading protein-A affinity chromatography to maximize
utilization and reduce cost of operation
Alejandro Becerra-Arteaga, alejandro.becerra-arteaga@
merckgroup.com, Bala Raghunath.Biomanufacturing Sciences Network,
EMD Millipore, Billerica, MA 01821, United States
Protein A media are one of the major contributors in the cost of
production of monoclonal antibodies particularly during clinical
phase. A conservative safety factor (e.g. 80% of 10% breakthrough)
is generally used during the loading step in this unit operation.
Here we analyze an alternative approach of loading to 1-5%
breakthrough and the effect on process economics and productivity.
A comparison between resins having different breakthrough curve
slopes as well as implementation and scale up considerations are
also discussed.
BIOT 435
Performance of different Protein A resins with respect to
critical attributes
Jelena Vasic, [email protected], Anders Ljunglöv, Ronnie Palmgren,
Tomas Björkman, Karol Lacki.GE Healthcare, Uppsala, Sweden
Protein A affinity chromatography is the major purification
technique for capture of antibodies and enables a platform approach
to downstream processing. Currently there are several Protein A
resins available on the market. The resins vary in their different
properties, including material of base matrix, type of Protein
A ligand, particle and pore sizes. As a results chromatographic
attributes of these resins, such as binding capacities, elution pH
and purity of eluted antibody can vary. In addition, process related
attributes such as packability of the resin in large scale columns
and resin life time will also be different. These variations in resin
performance leave a process development scientist with a need to
compare different resins and to assess their potential advantages.
In this study we have compared five Protein A resins by looking at
typical performance attributes such as dynamic binding capacity
at different residence times and resins life time (using real feed
and buffer solutions). We have also evaluated purity of the eluted
pools by looking at leached Protein A and HCP levels. The results
obtained allow ranking of the resins according to different process
scenarios.
BIOT 436
HA displayed yeast influenza vaccine
BIOT 434
Purification of antibody fragments: A new route for capture
Carina Engstrand, [email protected], Björn Norén, Bengt
Westerlund, Peter Lindahl, David Westman.Chemistry, GE Healthcare,
Uppsala, Uppsala 75184, Sweden
Different variants of classic monovalent antibody fragments
(Fab, scFv Dab etc.) are now emerging as credible alternatives to
monoclonal antibodies (mAbs). There are several different types
of antibody fragments that can retain the targeting specificity of a
full antibody but lack the Fc-portion.
The common way to purify monoclonal antibodies is by a platform
approach using affinity chromatography with Protein A as the
capture step. The high purification factor and generic conditions
used with this approach outnumbers price and low alkaline stability
of the proteinaceous ligand. However, for antibody fragments
lacking the Fc region there has not yet been a generic solution. Here
we present a potential generic capture step for antibody fragments
of different sizes. Features of this new affinity chromatography
media are presented along with application examples.
Lu Zhang, [email protected], Qinglong Liang, Yarina Masniuk, Sha
Jin, Kaiming Ye.Biomedical Engineering Program, College of Engineering,
University of Arkansas, Fayetteville, Arkansas 72701, United States
To date,vaccine has still been most effective for preventing both
seasonal and pandemic flu. Although traditional egg-based
influenza vaccine production has been proven effective, it suffers
from several drawbacks such as a long production period. Here
we present a new technology for rapid and massive influenza
vaccines production. The core of this technology is based on a
yeast viral antigen display system. These new vaccines are safer
than viral vector-based vaccines for use in humans. Our previous
work showed that yeast can effectively express functional H5N1
hemagglutinin (HA), and surface-displayed yeast vaccines can
elicit protective immune responses against influenza in mice. In
this work, we further optimize the expression system in order
to enhance the immunogenicity of the yeast surface displayed
viral antigens. Animal experiments are planned to determine the
effectiveness of these new vaccines. The results will be presented at
the conference.
159
Wednesday Afternoon
BIOT 429
Purification of antibody fragments: A new route for capture
Carina Engstrand, [email protected], Björn Norén, Bengt
Westerlund, Peter Lindahl, David Westman.Chemistry, GE Healthcare,
Uppsala, Uppsala 75184, Sweden
Different variants of classic monovalent antibody fragments
(Fab, scFv Dab etc.) are now emerging as credible alternatives to
monoclonal antibodies (mAbs). There are several different types
of antibody fragments that can retain the targeting specificity of a
full antibody but lack the Fc-portion.
The common way to purify monoclonal antibodies is by a platform
approach using affinity chromatography with Protein A as the
capture step. The high purification factor and generic conditions
used with this approach outnumbers price and low alkaline stability
of the proteinaceous ligand. However, for antibody fragments
lacking the Fc region there has not yet been a generic solution. Here
we present a potential generic capture step for antibody fragments
of different sizes. Features of this new affinity chromatography
media are presented along with application examples.
microarray datasets.
Given the state of gene switches governs the phenotype, we
postulate that recognizing specific gene switches would enable
the identification of molecular signatures that would be better
drug targets for treating a disease. We demonstrate the utility
of our mining approach with human breast cancer by analyzing
a paired breast cancer-normal tissue expression dataset against
the integrated human gene expression dataset. A list of genes
predicted to be gene switches for breast cancer include known
targets, e.g. estrogen receptor and Her2 gene. In addition to these
known targets, the approach also discovered novel targets such as
TACSTD2. TACSTD2 is predicted to be an important biomarker
for both estrogen (ER)+ and ER– breast cancer subtypes, and
therefore is an attractive candidate for treating different subtypes of
breast cancer. We predict through annotation, sequence matching
of TF sites, and TF activity estimation, a novel transcriptional
mechanisms by which TACSTD2 is regulated. Our experiments in
breast cancer cell lines confirm the functional role of this gene in
breast cancer and for the first time identify a likely transcription
factor involved.
BIOT 438
BIOT 437
Integrative analysis of transcriptome identifies gene switches
as novel biomarkers and potential targets for human breast
cancer
Ming Wu1, [email protected], Li Liu4, [email protected], Christina
Chan1,2,3, [email protected]. (1) Department of Computer Science
and Engineering, Michigan State University, East Lansing, Michigan 48824,
United States (2) Department of Chemical Engineering, Michigan State
University, East Lansing, Michigan 48824, United States (3) Department
of Biochemistry and Molecular Biology, Michigan State University, East
Lansing, Michigan 48824, United States (4) Department of Microbiology
and Molecular Genetics, Michigan State University, East Lansing, Michigan
48824, United States
Gene switches, or genetic switches play central roles in cell fate
decision, and their dynamic property of “bistability” ensures
that a graded signal is converted into a binary response, which
unequivocally commits the cells to a specific phenotype. As
an emergent property that arises from regulatory interactions,
it is difficult to identify switches without a priori analysis of
the network. Therefore a challenge confronting the field is to
systematically identify gene switches on a genome scale. We
propose a top-down mining approach to identify gene switches
from gene expression data, wherein microarray data integrated
across many different conditions provide a large sampling of the
transcriptome state space. We perform a theoretical analysis and
provide proof-of-concept applications on both synthetic and yeast
160
Quantitative Structure Activity Relationship (QSAR) models
for the lead optimization of antibody and peptide affinity
reagents
James A Woo1, [email protected], Divya Chandra1, Pankaj Karande1,
Sachdev Sidhu2, Steven M Cramer1. (1) Department of Chemical and
Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York
12180, United States (2) Banting and Best Department of Medical
BIOT 439
BIOT 441
Probing protein selectivity in hydroxyapatite using single
molecule atomic force microscopy
Using simulation for bioprocess technology transfer and
process-facility fit
Kartik Srinivasan, [email protected], Steven M Cramer.Department of
Demetri Petrides, [email protected], Charles Siletti.
Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy,
New York 12180, United States
Studies have shown that ceramic hydroxyapatite (CHA) combines
multiple modes of interactions to create unique selectivity windows
for protein separations in the presence of different displacers
and mobile phase modifiers. In the present work, atomic force
microcopy (AFM) is employed to gain fundamental insights into
the principles that govern such selective behavior. Single molecule
AFM is used to probe the energetics of protein interaction with
CHA by functionalizing the AFM probe with a protein of interest
and performing force-distance measurements. The free energy
of binding and force of interaction between the immobilized
protein and the CHA surface are then investigated in the presence
of different mobile phase modifiers and displacers. The relative
binding strengths obtained from these experiments are validated
qualitatively against column chromatographic retention data. The
insights gained from these studies are used to reveal the nature of
the selectivity of CHA for protein separations.
BIOT 440
Production planning, scheduling, and debottlenecking
practices in the biopharmaceutical industries
Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada
Charles Siletti, [email protected], Demetri Petrides, dpetrides@
The a-priori prediction of binding affinity is important for
applications ranging from therapeutics to biosensors and
separations. In this poster,Quantitative Structure Activity
Relationship (QSAR) models were developed for both antibody
and peptide affinity systems. Models were designed topredict the
affinities of antibody fragments bound to the HER2 antigenfrom
the sequences of their CDR loops. Using 3D molecular
descriptorsdescribing the geometry, charge and hydrophobicity
of these loops,accurate and predictive models (R2 > 0.95) were
generated. QSAR modelswere also developed using novel sequencebased descriptors to predict the binding affinity of peptides to
human transferrin and S-protein. Thesedescriptors systematically
compare the difference in physicochemicalproperties between any
two residues in a peptide, identifying keypositions in the sequence
and providing insights into the desirablephysiochemical properties
for that position. This work can providevaluable guidance in the
lead optimization process for developing newaffinity reagents.
This paper presents industrial experience with a resourceconstrained batch process scheduling tool. The scheduling
algorithm is a non-optimization approach that proceeds in two
steps. First a bottleneck analysis is done to determine a lower
bound on the process cycle time, and all the batches are scheduled
accordingly. Second, if conflicts remain, they are resolved by
applying progressively aggressive modifications to the schedule.
This approach to scheduling was tested on several biotech
processes. The scheduling challenges in biotech processes lie in
the ancillary operations: media and buffer preparation, vessel and
line cleaning, and chromatography column preparation. Such
operations may use shared resources that can to couple process
suites with otherwise dedicated equipment. Three case studies,
which are based on a process for the manufacture of monoclonal
antibodies (MABs), illustrate the value of a constrained-resource
scheduling tool for biotech processes.
intelligen.com.Intelligen, Inc., United States
Intelligen, Inc., Scotch Plains, NJ 07076, United States
Technology transfer is, in large part, transfer of process knowledge
from the development group to a manufacturing site. Because
process simulation models represent a succinct, functional
repository of process knowledge, they are a natural addition to the
technology transfer process. This paper focuses on the capabilities
and limitations of simulation models for technology transfer.
Process-Facility fit is part of the technology-transfer process and
is ultimately a determination of whether and how well a given
process can be executed in a particular facility. Gap analysis is often
the technique of choice for evaluating a facility. This presentation
primarily focuses on the use of process simulation and scheduling
tools to add quantitative information to gap analysis. A series of
examples demonstrate how simulation can aid in determining gaps
in primary equipment, buffer preparation equipment, purified
water, utilities, labor and staffing. Techniques for managing
uncertainties in process facility fit will also be covered.
BIOT 442
Scale-down model qualification methodology for a monoclonal
antibody program
Uma Balasubramanian, [email protected], John
Easson, Ayushman Ghosh, Gargi Maheshwari, Peter Russo.Department
of Biologics Manufacturing Sciences and Commercialization, Merck and
Company, Rahway, NJ 08852, United States
The use of scale-down models for bioprocess unit operations is
critical for any program that aims to rapidly meet the challenges
of commercialization. Scale-down models are commonly used
for process development, process characterization and lab-scale
troubleshooting of large-scale commercial processes. With line
of sight to manufacturing facilities and equipment, a scale-down
model checklist was developed that allowed the identification of
scale-dependent and scale-independent parameters along with
gaps to the commercial process. We will present a case study in
which a 3-L bioreactor was established as a scale-down model to the
5000-L manufacturing scale for a therapeutic monoclonal antibody
process. Our methodology toward scale-down qualification of the
3-L bioreactor and its use as a predictive and directional indicator
of manufacturing scale will be discussed. The success of this model
will be assessed by the analysis of process outputs, such as peak cell
density, metabolic profile, product titer and quality.
161
Wednesday Afternoon
BIOT 436
BIOT 445
3D lithographically structured cell culture devices
Automated molecular diagnostic system for personal medicine
based on magnetic separation
Yevgeniy V Kalinin, Jaehyun Park, Christina L Randall, David H
Gracias, [email protected] and Biomolecular Engineering, The
Johns Hopkins University, United States
We describe the utilization of lithographic processes to precisely
structure cell culture devices from the nano to the macro scale.
Using a combination of theoretical modelling and experimentation,
we describe utility of these devices for in vitro cell culture and
designing bio-artificial organs. Devices feature porosity in all three
dimensions to enhance nutrient diffusion and reduce hypoxic
zones. Nanoporosity also offers the possibility for immunoisolation
of implanted cells. The devices are also precisely structured on
the micro and macro scale to enable adequate diffusion and high
precision with respect to size, shape and array spacing, which can be
achieved on both rigid and flexible substrates. Our results suggest a
new and precise synthesis paradigm for cell culture devices.
BIOT 444
Establishment of new logic for the analysis of proeins based on
properties of amino acids
Chaeul Ku2, [email protected], Hyobong Hong1, Yongjoon Lee1. (1)
Dept. of Fusion Technology, ETRI, Daejeon, Choongnam 305-700, Republic
of Korea (2) TurboSoft Co., Chungwon gun, Chungbuk, Republic of Korea
In this study, hemagglutinin proteins of several influenza A virus
and related proteins were employed as the model compounds. The
physico and chemical properties employed in this research are
hydropath index, Log P, Gibbs Free Energy, tPSA and numbers
of donors (or acceptors) of hydrogen bonds. After converting the
each amino acid to the physico/chemical properties based on the
previous researches, matrix consisting of the amino sequences
and the properties of the amino acids was prepared. Then, the
proteins were characterized and categorized based on the physico/
chemical properties of amino acids not based on the alignment
of the sequences. The results indicated that influenza viruses
could be categorized and characterized by the new methods. For
example, average of the hydropath index and Log P values of the
influenza viruses are in the certain values (-1.32~1.48) and the
values are different from those of another non-hemagglutinin
proteins employed in this research. These results showed that our
approaches can be the alternatives of the existed ones and the new
way to categorize the proteins.
162
Bin Liu, Song Li, Hongna Liu, Nongyue He, [email protected]
of Biological Science and Medical Engineering, State Key Laboratory of
Bioelectronics,Southeast University, Nanjing, Jiangsu 210096, China
This article puts nano-technology, biological chip technology
and magnetic particles array technology together, and develops
the molecular diagnosis method which fits individual medical
treatment demand. Meanwhile, it combines the unique fully
enclosed detector design with signal scanners, constructing
automated molecular diagnostic system for personal medicine
based on multiplex PCR with fully enclosed, automation and
high practical. This system takes 96-well PCR plate as the reaction
carrier, including mechanical arm, heating and cooling platform,
fluorescence detection platform etc. It can realize the whole process
automatically from genomic DNA extraction, target sequence
amplification, hybridization to fluorescent signal detection. It
breaks through the technical bottleneck of the traditional multiplex
PCR technique technologies, with high sensitivity, specificity and
high yield characteristics, and it is applicable to the high throughput
analysis and diagnosis.
BIOT 446
Integrated upstream and downstream strategy to control the
level of acidic variants and improve downstream productivity
for a monoclonal antibody
Sandra Meissner1, [email protected], Thomas Linden1,
Joseph Nti-gyabaah1, Edward Glowacki1, Patricia Rose3, Rebecca
Chmielowski1, Janelle Konietzko2. (1) Department of Protein Purification
Development, Merck&Co, Inc, Union, NJ 07083, United States (2)
Department of Vaccine Process Development, Merck&Co, Inc, West Point,
PA 19486, United States (3) Department of Fermentation Development,
Merck&Co, Inc, Union, NJ 07083, United States
Formation of acidic antibody variants can occur at any point
during product manufacturing or product storage, and may affect
product efficacy and antibody stability. Our protein of interest
(POI) was found to contain inherently high levels of acidic variants,
due to its multiple deamidation sites. High throughput screening
(HTS) and design of experiment (DoE) showed that designing a
purification scheme to clear acidic variants once generated would
be cost-prohibitive, and the resulting impact on process yield
would be consequential. As an alternative, parameters such as cell
culture duration in conjunction with harvest and product storage
conditions, as well as the early purification steps were optimized to
minimize the rate of variant formation. This poster presents data
around the integrated upstream and early purification efforts that
led to an effective control of acidic variant formation. Acceptable
variant levels in the final drug substance were achieved without any
effect on process yield.
BIOT 447
Effect of ionic strength and drug payload on the stability of
antibody drug conjugates
Yilma T Adem, [email protected], Kelly Schwarz, Osigwe Esue.
Early Stage Pharmaceutical Development, Genentech, South San Francisco,
CA 94080, United States
Antibody drug conjugates (ADC) contain a monoclonal antibody
attached to a potent cytotoxic agent through a protease labile linker.
ADCs are anticipated to have significant advantage over standard
chemotherapy agents as they have a potential to be specific to the
target and highly effective with lower toxicity. In recent years,
ADCs were getting considerable attention by drug developers as
potential treatment options for various types of cancers. During
the formulation development of Mab-A, substantial amount of
aggregation was observed when Mab-A was formulated in high
ionic strength buffers. We investigated the cause and found that the
high drug load species of Mab-A were more prone to aggregation
than the low drug load species.
BIOT 448
and higher resolution protein structures. These results support
our hypothesis that chiral interactions are important in protein
crystallization.
BIOT 449
Applications of particle sizing techniques in mammalian cell
culture harvest process development
Krista Petty, [email protected], Xiaoyang Zhao, Junfen Ma, Tim
Tressel.Purification Process Development, Amgen Inc., Newbury Park, CA
91320, United States
Advances in mammalian cell culture processes have necessitated
the development of harvest technologies that are capable of
clarifying cell culture feed stocks with high cell densities or
solid contents. This has required improved understanding and
utilization of fundamental separation principles. The speed and
efficiency of harvest processes are influenced by a number of factors
including the nominal particle size and particle size distribution
of the suspended solids. The size of the suspended solids can be
modulated through pre-harvest additions, such as flocculants, or
by processing conditions, such as mixing or pumping. Thus, in
optimizing harvest processes, it is critical to understand the factors
that change the suspended solids particle size.
The use of commercially available particle sizing technologies,
including the Focused Beam Reflectance Measurement technology,
in the development of a flocculant harvest process will be discussed.
Applications of particle sizing data to predict optimal flocculant
dosing and depth filtration capacity will also be discussed.
Effect of chiral additives on protein crystallization
Mark Stauber1, [email protected], Jean Jakoncic2, Jacob Berger1, Ariel
Axelbaum1, Jerome Karp1, Neer Asherie1. (1) Department of Physics and
Department of Biology, Yeshiva University, New York, New York 10033,
United States (2) National Synchrotron Light Source, Brookhaven National
Laboratory, Upton, New York 11973, United States
Chiral additives have proven useful to control the crystallization of
small molecules, but the role of chirality in protein crystallization
has yet to be investigated systematically. Previously, we discovered
that the chirality of sodium tartrate affects the crystallization
of thaumatin. For example, L- and D-tartrate produce different
crystal habits due to a preferential interaction between thaumatin
and the L enantiomer. To establish the generality of our findings,
we are studying other protein-precipitant pairs. Here we present
our results for lysozyme and 2-methyl-2,4-pentanediol (MPD); the
racemic (RS) mixture of MPD is one of the most commonly used
additives in protein crystallization. We successfully crystallized
lysozyme with enantiomerically pure R-MPD and S-MPD as well
as with RS-MPD. We find that there is a preferential interaction
between lysozyme and R-MPD producing more ordered crystals
BIOT 450
Protein adsorption and selectivity in multimodal
chromatographic systems
Melissa A Holstein1, [email protected], Siddharth Parimal1, Scott
A McCallum2, Steven M Cramer1. (1) Department of Chemical and
Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York
12180, United States (2) Department of Biology, Rensselaer Polytechnic
Institute, Troy, New York 12180, United States
While the unique selectivities, broad applicability, and enhanced
separation power of multimodal chromatographic resins have
resulted in more widespread use of these materials in recent years,
the separation of proteins with similar properties and retention
behavior continues to be a challenging problem. Chromatography,
nuclear magnetic resonance, and molecular dynamics simulations
are used to provide fundamental insight into protein adsorption
in multimodal chromatographic systems. Column experiments
and high-throughput batch chromatography experiments are
163
Wednesday Afternoon
BIOT 443
BIOT 451
Removal of the genotoxic impurities acetamide and
thioacetamde from pharmaceutical formulations using a
hybrid approach
Emelie Fritz1, [email protected], Elin Rundquist2,
Börje Sellergren1. (1) Institute of Environmental Research (INFU) of
the faculty of Chemistry, Technical University of Dortmund, Dortmund,
NW 44227, Germany (2) Particle Generation, Control & Engineering,
GlaxoSmithKline Ltd., Stevenage, United Kingdom
The synthesis of pharmaceutical products often involves the use
of reactive reagents for the formation of intermediates and APIs.
Low levels of reagents or by-products may therefore be present in
the final drug product as impurities. Some of these impurities are
chemically reactive and may have unwanted toxicities, including
genotoxicity and carcinogenicity, with severe impact on product
risk assessment. Recently GTIs have gained increased attention
and pharmaceutical regulatory authorities carry on issuing
guidelines that strict the limits of genotoxins in medicines1. In this
EU project2 new purification techniques for the pharmaceutical
industry with molecularly imprinted polymers in different formats,
e.g. membranes and thin films are developed. Here we report on
the synthesis, chromatographic characterisation and removal
test, as well as assessment of economical and process application,
of the first generation of such materials targeting acetamide and
thioacetamide.
1 EMEA/CHMP/QWP/251344/200
2 NEMOPUR www3.imperial.ac.uk/molecularpurification (Nr.
PITN-GA-2008-214226)
BIOT 452
Study of the mechanisms of antibody aggregation in solution
Frida Ojala1, [email protected], Marcus Degerman1, Thomas
Budde Hansen2, Ernst Broberg Hansen2, Bernt Nilsson1. (1) Department
of Chemical Engineering, Lund University, Lund, Sweden (2) Novo Nordisk
A/S, Bagsværd, Denmark
In order to avoid aggregation during preparative separation of
antibodies, it is desirable to have thorough knowledge of the
mechanistic behavior of the reaction. In this work a study of antibody
164
aggregation occurring in a solution has been conducted. The main
aim was to find a methodology to determine the mechanisms of
the reaction and to find the dependence of the kinetic parameters
on the experimental conditions. The aggregation reaction can be
sensitive to antibody and salt concentration as well as pH and
temperature. Samples with varying conditions has been analysed
with SEC at different time points to produce a time dependent
model of the system. Calibrations to different reaction models
were then conducted, setting the kinetic parameters and finding
the likely mechanism. The trends in the parameters achieved were
then evaluated based on the initial conditions studied. The reaction
parameters proved highly dependent on both salt concentration
and temperature.
BIOT 453
New photoelectric instrument for dual-color fluorescence
detection
Zheng Liu, Yun Xia, Bin Liu, Qihua Li, Nongyue He, nyhe1958@163.
com.Department of Biological Science and Medical Engineering, Southeast
University, Nangjing, Jiangsu 210096, China
Generally the photoelectric detection system contains optical
construction, mechanical parts processing, signal detection
and follow-up circuit processing. This instrument consists of
halogen tungsten lamp source, dichroscope, filter, lens and
photomultiplier to achieve dual-color fluorescence detection
based on Cy3 and Cy5. The instrument achieved transmission of
excited light and acquisition of emission light to detect samples
with high sensitivity by ingenious construction. Based on the
characteristics of photomultiplier, the signal detection section
includes matched filtering amplification, AD conversion circuit
and control unit to ensure high SNR (Signal to Noise Ratio) and
accuracy. The tailored make of related mechanical components
ensures reliable and stable fluorescence detection of the whole
instrument. Through multiple measurements of samples, it can be
confirmed that the instrument can preferably distinguish different
fluorescence intensity to satisfy detection sensitivity requirements
of hospitals and laboratories.
BIOT 454
Biomacromolecule detection system based on magnetic
separation
Bobo Zhang, Nongyue He, [email protected], Bin Liu, Zheng Liu,
Hui Chen.School of Biological Science and Medical Engineering, State Key
Laboratory of Bioelectronics,Southeast University, NanJing, Jiangsu 210096,
China
Biomacromolecule detection system aims to develop a kind of
automation analysis system, which is fast, high-throughput and can
be applied to clinical diagnosis. Using 96-well plate as the carrier,
this system mainly consists of a magnetic separation module, a
high accurate Heating and Cooling module, a mechanical moving
module, an accurate liquid transfer module, a fluorescent detection
module and a reagent storage station.
Utilizing all these modules, the automatic processing of samples in
the plate can be realized. By combining with functional magnetic
nanoparticles, high sensitivity fluorescence detection technology,
and related biochemical analysis techniques, biomacromolecule
detection system can be used in gaining various biomacromolecule
information , SNP sites analysis, nucleic acid mutation detection,
immune analysis and large-scale blood census test. The development
of this system would perform as a strong instrument in the study of
science research and human health.
BIOT 455
fragmentation method on a linear ion trap mass spectrometer.
We conclude that DiART reagents can be used to accurately and
reproducibly perform quantitative proteomic analysis using PQD
mode of fragmentation at a significantly lower cost.
BIOT 456
Purification of peptide from antibody conjugates using anion
exchange membrane chromatography
Erin Kinney, [email protected], Brandi R Osborne.Biological
Research and Development, Pfizer, Chesterfield, Missouri 63017, United
States
Anion exchange membrane chromatography is a promising
alternative purification method to traditional columns for removal
of contaminants from proteins and antibodies, and has typically
been applied for removal of trace contaminants such as DNA,
HCP, and endotoxin. This method can also be applied during the
purification of bioconjugate therapeutics and vaccines, which are
increasingly prevalent in the biopharmaceutical industry. Anion
exchange membrane chromatography provides a simple alternative
for removal of residual reaction components, such as unreacted
peptides and linkers. The residual reaction components following
a conjugation reaction can make-up a significant amount of the
feed stream (2-5%). Demonstration of this technique for removal
of unreacted peptide from a number of antibody conjugates will
Applicability of novel and cost effective 6-plex isobaric tagging
reagent, DiART using model protein mixtures on a linear ion
trap (LTQ Xl) mass spectrometer
Nikhil Ramsubramaniam1, [email protected], Feng Tao2, ftao@
omicbiosystems.com, Mark Marten1, [email protected]. (1) Chemical
and Biochemical Enginering, University of Mayland Baltimore County,
Baltimore, Maryland 21250, United States (2) Omic Biosystems, Rockville,
Maryland 20850, United States
DiART is an isobaric, peptide mass-tagging reagent which
facilitates relative protein quantitation in a functionally similar
manner to well established iTRAQ and TMT reagents. Unlike
iTRAQ and TMT reagents, DiART reagents harbor deuterium
isotopes. Incorporating deuterium, instead of carbon and nitrogen
isotopes, significantly reduces the number of synthesis steps and
hence reduces the cost of these reagents. By carefully controlling
the position and the number of deuterium labels, DiART reagents
have been shown to be devoid of deleterious isotope effects like
chromatographic shifts associated with deuterium labels during
reverse phase chromatography. Here, we test the applicability of
DiART reagents using model protein mixtures and complex fungal
cell-wall protein extract using a pulsed Q dissociation (PQD)
be described including examples of the challenges encountered
during development of these peptide removal steps.
165
Wednesday Afternoon
carried out with a variety of mobile phase modifiers. These
experimental techniques are used in conjunction with molecular
dynamic simulations to understand protein-ligand interactions
at the molecular level and to characterize the electrostatic,
hydrophobic, and synergistic interactions present in multimodal
chromatographic systems.
Nanomechanical properties of cartilage
Ferenc Horkay1, [email protected], Iren Horkayne-Szakaly1,
Emilios K. Dimitriadis2, Candida Silva1, Peter J. Basser1. (1) NICHD,
National Institutes of Health, Bethesda, Maryland 20892, United States (2)
NIBIB, National Institutes of Health, Bethesda, Maryland 20892, United
States
The mechanical properties of cartilage strongly depend on the
composition of the extracellular matrix, in which negatively
charged proteoglycan (PG) molecules are enmeshed in a collagen
network. The collagen provides tensile strength while the highly
charged anionic PGs create an osmotic swelling pressure. The
structure of the cartilage matrix and the distribution of the
components are strongly depth-dependent. The PG content is
lowest in the superficial zone and highest in the middle zone. The
fixed charge density is highest in the deep zone. We determined the
depth dependence of the elastic and osmotic modulus of cartilage
using a novel combination of atomic force microscopy (AFM) with
osmotic swelling pressure measurements. AFM indentations detect
fine spatial variations in the mechanical properties of cartilage
layers. This study also reveals that the osmotic modulus of articular
cartilage is highest in the superficial and deep zones, and lowest in
the middle zone.
BIOT 458
AAPH induced mAb oxidation and aggregation
Kai Zheng1, [email protected], Diya Ren2, Wayne Lilyestrom1,
Yatin Gokarn1, Robert Bayer2, Thomas Scherer1, Y. John Wang1, Junyan
A. Ji1. (1) Late Stage Pharmaceutical Development, Genentech, South
San Francisco, California 94080, United States (2) Oceanside Pharma
Technical Development, Genentech, Oceanside, California 92056, United
States
Oxidation is a key degradation pathway in protein drugs. During
a therapeutic antibody formulation development, oxidation
stress was induced by a free radical generator, 2,2’-Azobis
(2-amidinopropane) dihydrochloride (AAPH). In addition to
methionine and tryptophan oxidation, we observed the aggregation
increase. Size-exclusion chromatography and multi-angle light
scattering (SEC-MALS) showed that high molecular weight species
(HMWS) contained dimer, tetramer, and higher order aggregates.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) showed that the newly formed HMWS was mainly
covalently linked and intermolecular disulfide cross-linking was
a major contributing factor. Tryptic map indicated methionine
and tryptophan oxidation, but did not reveal any non-disulfide
cross-linking sites. Fluorescence spectra suggested that bityrosine
166
may contribute to cross-links. Additional study indicated that
tryptophan, tyrosine, or pyridoxine could protect antibodies’
aggregate formation from AAPH stress.
In summary, our data exhibit the complicated mAb oxidation
products under chemical stress and mAb oxidation should be
closely monitored during biotherapeutic development.
BIOT 459
Measuring antibody drug conjugate positional isomers
William J Galush, [email protected], Lan N Le, Jamie MR
Moore.Early Stage Pharmaceutical Development, Genentech, Inc., South
San Francisco, CA 94080, United States
Antibody drug conjugates enable the targeted delivery of potent
chemotherapeutic agents directly to malignant cells. They are
made by the chemical conjugation of cytotoxins to monoclonal
antibodies, which can be achieved by first reducing interchain
disulfide bonds followed by conjugation with drugs. This process
yields a controlled, but heterogeneous, mixture of conjugated
products, which contains species of various drug-to-antibody
ratios as well as different positional distributions of drugs on
the antibodies. We have developed a mathematical algorithm
using inputs from capillary electrophoresis and hydrophobic
interaction chromatography to determine the positional isomer
distribution within a sample. The procedure is amenable to rapid
sample analysis and features low material requirements. Using this
technique, a survey of several antibody drug conjugates has shown
a very similar distribution of isomers among all of the molecules,
suggesting a robust conjugation process.
BIOT 460
Physicochemical and thermodynamic properties of tea and soy
polyphenols
David Mendez Sevillano1, [email protected],
Luuk A.M. van der Wielen1, Marcel Ottens1, Olivera Trifunovic2. (1)
Department of Biotechnology, TU Delft, Delft, Zuid Holland 2628BC, The
Netherlands (2) Unilever R&D, Vlaardingen, Zuid Holland 3130AC, The
Netherlands
Nutraceuticals are food constituents with a positive effect on
health. These compounds are present in food and beverages at
low concentration with complex interactions with the solid matrix
of plant materials. Isolating these components in large scale
production requires knowledge of their physical-chemical and
thermodynamic properties.
In this paper, antioxidants of the class of polyphenols are
investigated. These polyphenols can be found in tea (catechins
in green tea, theaflavins in black tea) and in soy (isoflavones).
This study aims at measuring and predicting solubility of these
polyphenols in different solvents.
This paper presents activity coefficient models that are specifically
developed to predict this solubility with input of the mixture
composition and temperature plus the implementation of a high
throughput solubility experimental methodology developed
to measure the interaction between the pure components and
the different solvents (and mixtures). The developed model can
accurately predict the solubility of polyphenols in different solvents.
BIOT 461
materials is a challenging topic, strong binding between designed
synthetic materials and specific proteins and peptides has been
reported in several successful examples. Previously, we reported
designed synthetic polymer NPs (∼30 nm) capable of binding
melittin[1-4], a 26 amino acid peptide toxin isolated from bee
venom. Recently, we have expanded our targets to include proteins.
NP-protein binding takes advantage of the unique presentations of
functionality on the protein surface. Designed synthetic polymer
NPs were able to capture a target protein from a mixture of proteins.
[1] J. Am. Chem. Soc., 2008, 130, 15242–15243.
[2] Small, 2009, 5, 1562–1568.
[3] J. Am. Chem. Soc., 2010, 132, 6644-6645.
[4] J. Am. Chem. Soc., 2010, 132, 13648-13650.
Aerosolized bacterial challenge of disposable aseptic
connectors used in bioprocessing applications
BIOT 463
Jonathan Royce1, [email protected], Alisa Liten2, alisa.liten@
ge.com. (1) Bioprocess Division, GE Healthcare Life Sciences, Uppsala,
Sweden (2) Bioprocess Division, GE Healthcare Life Sciences, Piscataway,
NJ 08854, United States
Disposable aseptic connectors (DACs) are becoming standard
connection tools for single-use bioprocess manufacturing. DACs
enable single-use processing equipment and unit operations to be
safely and quickly connected while minimizing or even eliminating
the need for classified manufacturing space. Currently, there are
no regulatory standards which define the level to which DACs
must maintain sterility in non-sterile environments. Therefore,
manufacturers are expected to provide regulatory support material
to end users. This poster explains recent work performed by GE
Healthcare Life Sciences to expand the material available for
ReadyMate disposable aseptic connectors. An aerosolized microbial
challenge was used to verify the ability of ReadyMate connectors to
maintain sterility under extreme conditions. Transfers of culture
medium were performed after each connection and the transferred
medium was incubated to verify sterility. Positive and negative
controls were included in the study. A 100% success rate was
achieved.
Thermodynamics of the molecular interactions between
amyloid β-peptide fragments and (-)-epigallocatechin-3gallate
Shihui Wang, Xiaoyan Dong, [email protected], Yan Sun, ysun@tju.
edu.cn.Department of Biochemical Engineering, Tianjin University, Tianjin,
China
BIOT 462
(-)-Epigallocatechin-3-gallate (EGCG) has been proved effective in
preventing the aggregation of amyloid β-peptide 42 (Aβ42), and
the thermodynamic interactions between them were studied in our
previous work. To further probe the interactions between different
regions of Aβ42 and EGCG, three Aβ42 fragments (Aβ1-16, Aβ130, and Aβ31-42) were synthesized and the interactions between
each of the fragments and EGCG were investigated by isothermal
titration calorimetry. It is found that though hydrogen bonding
and hydrophobic interaction are both involved in the interactions
between Aβ42 and EGCG, hydrogen bonding mainly happens in
Aβ1-16 while hydrophobic interaction mainly happens in Aβ1742. When Aβ42 and its fragments are saturated by EGCG, the
thermodynamic parameters for them have linear relationships. It
suggests that there are not specific interactions and binding sites
in the Aβ42 and EGCG binding. Moreover, there is significant
enthalpy-entropy compensation in the binding of EGCG to Aβ42
and its fragments.
Selective protein capture using designed synthetic polymer
nanoparticles
BIOT 464 - Withdrawn
Keiichi Yoshimatsu, [email protected], Benjamin K Lesel,
Yu Hoshino, Kenneth J Shea.Department of Chemistry, University of
California, Irvine, Irvine, CA 92697, United States
Synthetic nanopartciles (NPs) capable of binding to proteins
are attracting attention due to their significant potential in
biotechnology. Although selective protein binding by synthetic
167
Wednesday Afternoon
BIOT 457
Methods for reduction of water flux rates and volumes for
extractables removal from cellulosic depth filters
George Oulundsen, [email protected], Seat Yee
Lau, Domenico Origi, Michael Felo.Biomanufacturing Sciences Network,
EMD-Millipore, Billerica, MA 01821, United States
Cellulosic depth filters are commonly used in many steps during
the production of biopharmaceuticals including bioreactor harvest,
low pH viral inactivation, and virus filtration. At production scale,
the need to flush these filters before use can place a significant
burden on purified water supply and pump capacity. The volume of
costly WFI and the flowrates recommended by vendors are often
difficult to achieve and implement.
The effect of reduced flux rates on the removal of extractables has
been examined for depth filters used for primary and secondary
clarification. Methods for reduction of water consumption such as
flushing multiple filters in series and recirculation of flush water
were investigated. Results indicate that a 50% reduction in flux
rates from the manufacturer’s recommendation provides sufficient
removal of extractables as measured by conductivity and TOC
levels. Flushing of depth filters in series reduced the total WFI
volume required by at least 50%, while recirculation of flush water
can reduce WFI volume by 30-50%. Reduction in flush water flux
rates and volumes facilitates the implementation of these effective
clarification devices in biopharmaceutical processes.
BIOT 466
Single-molecule recognition of biomolecular interaction based
on Kelvin probe force microscopy
Taeyun Kwon1, [email protected], Kilho Eom2, kilhoeom@
gmail.com, Dae Sung Yoon1, [email protected], Jaemoon Yang3, Gyudo
Lee1, [email protected]. (1) Department of Biomedical Engineering,
Yonsei University, Wonju, Kangwon-do 220-710, Republic of Korea (2)
Department of Mechanical Engineering, Korea University, Seoul, Republic
of Korea (3) Department of Radiology, College of Medicine, Yonsei
Unicersity, Seoul, Republic of Korea
168
We report the scanning probe microscopy (SPM)-based singlemolecule recognition of biomolecular interaction between protein
kinase and small molecule (e.g. ATP or imatinib). In general,
conventional atomic force microscopy (AFM) is unable to sense
and detect the small ligand bound to a protein kinase due to its
limited resolution for detecting the miniscule change in molecular
shape for protein kinase driven by ligand-binding. However, in
this study, we have first demonstrated that Kelvin probe force
microscopy (KPFM) enables the recognition of a single protein
kinase that interacts with a ligand (i.e. protein-ligand binding) at
single-molecule resolution due to its ability to measure a change in
the surface potential of a protein kinase induced by ligand-binding.
Moreover, the measured surface potentials of protein kinases
bound to ligands allows for quantitative understanding of ligandbinding mechanisms, which may be compared with theoretical/
computational predictions on ligand-binding. Our study sheds
light on KPFM that permits the precise recognition of singlemolecule interactions, which opens a new avenue for the design
and development of novel molecular therapeutics.
BIOT 467
AAPH induced mAb oxidation and aggregation
Kai Zheng1, [email protected], Diya Ren2, Wayne Lilyestrom1,
Yatin Gokarn1, Robert Bayer2, Thomas Scherer1, Y. John Wang1, Junyan
A. Ji1. (1) Late Stage Pharmaceutical Development, Genentech, South
San Francisco, California 94080, United States (2) Oceanside Pharma
Technical Development, Genentech, Oceanside, California 92056, United
States
Oxidation is a key degradation pathway in protein drugs. During
a therapeutic antibody formulation development, oxidation
stress was induced by a free radical generator, 2,2’-Azobis
(2-amidinopropane) dihydrochloride (AAPH). In addition to
methionine and tryptophan oxidation, we observed the aggregation
increase. Size-exclusion chromatography and multi-angle light
scattering (SEC-MALS) showed that high molecular weight species
(HMWS) contained dimer, tetramer, and higher order aggregates.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) showed that the newly formed HMWS was mainly
covalently linked and intermolecular disulfide cross-linking was
a major contributing factor. Tryptic map indicated methionine
and tryptophan oxidation, but did not reveal any non-disulfide
cross-linking sites. Fluorescence spectra suggested that bityrosine
may contribute to cross-links. Additional study indicated that
tryptophan, tyrosine, or pyridoxine could protect antibodies’
aggregate formation from AAPH stress.
In summary, our data exhibit the complicated mAb oxidation
products under chemical stress and mAb oxidation should be
closely monitored during biotherapeutic development.
BIOT 468
Suspended micron sized corner cube retroreflectors as
ultra-bright labels for pathogen diagnostics
Archana Kar1, [email protected], Tim Sherlock2, Azeem Nasrullah2, Julia
Litvinov3, Eliedonna Cacao1, Jennifer Knoop1, Steven Kemper1, Katerina
Kourentzi1, Paul Ruchhoeft2, Richard Willson1,4. (1) Department of
Chemical and Biomolecular Engineering, University of Houston, Houston,
TX 77204, United States (2) Department of Electrical and Computer
Engineering, University of Houston, Houston, TX 77204, United States (3)
Department of Biomedical Engineering, University of Houston, Houston,
TX 77204, United States (4) Department of Biology and Biochemistry,
University of Houston, Houston, TX 77204, United States
Corner cube retroreflectors (RR) are objects with three mutually
perpendicular reflective surfaces that return light directly to its
source and are therefore extremely bright and detectable using
simple, low-cost optics. In this work, we report the lithographic
fabrication of suspended five micron corner cube retroreflectors
and their use as ultra-bright labels in a rapid pathogen detection
assay. In preliminary experiments, the RR cubes were functionalized
with analyte specific antibodies and combined with the analyte (E.
coli bacteria) and magnetic sample preparation particles modified
with analyte-specific antibodies. We optically tracked the magnetic
“drag” of suspended cubes facilitated by analyte-driven binding
of magnetic particles onto the cubes. We further explore assay
integration with microfluidics for the development of a portable
and automatable diagnostic assay platform.
cell membrane are involved in this process, and we further study
altered phosphorylated kinase and associated pathways in the cell
upon Aβ addition.
BIOT 470
Molecular dynamics simulations of human islet amyloid
polypeptide (hIAPP) ion channels in lipid bilayers
Jun Zhao, [email protected], Xiang Yu, Chao Zhao, Qiuming Wang,
Jie Zheng.Department of Chemical and Biomolecular Engineering, The
University of Akron, Akron, OH 44325, United States
Amyloid ion channels have been postulated to disrupt the
permeability and integrity of cell membranes causing neuron cell
death, although the exact mechanism of amyloid ion channels
associated with ion conductivity and selectivity still remains
unclear. Here, we model and simulate a series of hIAPP ion
channels with different channel sizes (12-mer to 36-mer) and
structures (CNpNC and NCpCN) in DOPC bilayers to study the
conductivity and selectivity of ions, the structures and dynamics of
hIAPP channels and lipid bilayers. Molecular dynamics simulations
show that both CNpNC and NCpCN channels of varied sizes
are stable in the DOPC bilayers and exhibit poor selectivity for
cations, but CNpNC and NCpCN channels induce preferential Clpermeability across the bilayer in a complete opposite direction.
As compared to experimental results of poor selectivity for all
ions, a mechanism of hIAPP ion channels is proposed to bridge
simulation and experimental results.
BIOT 469
Deciphering Aβ interactions with the cell: Understanding
residue specific associations with the cell membrane, and
linking them to the changes in signaling pathways and synpatic
impairment observed in Alzheimer’s disease
Arundhathi K Venkatasubramaniam, [email protected], Theresa
Good.Chemical , Biochemical and Environmental Engineering, University
of Maryland Baltimore County, Baltimore, MD 21227, United States
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder that primarily affects the elderly population above 65 years
of age. The first signs of this disease are loss of learning and memory,
not death; hence we focus on understanding the mechanism of this
impaired synaptic function at a cellular level. Beta-amyloid peptide
(Aβ) in its aggregated form is believed to be toxic to neurons and
is implicated in AD. We seek to understand how Aβ interacts with
the cell, what signaling pathways it influences in the cell and how
these are related to the synaptic dysfunction and eventual necrosis
observed in AD. We investigate which residues of Aβ are involved
in its interaction with cell, whether particular receptors on the
BIOT 471
New approaches to immobilize cancer cells by using engineered
scaffold
Yu-Chen Lo1, [email protected], Hiroshi Honda2, Jorge Padmore2.
(1) Department of Biomedical Engineering, University of California, Los
Angeles, CA 90095, United States (2) Department of Bioengineering,
Northwestern Polytechnic University, Fremont, CA 94539, United States
A new approach to design a treatment procedure with minimum
side effects by using a engineered biodegradable scaffold to remove
and kill the cancer cells which could not be removed by surgery.
169
Wednesday Afternoon
BIOT 465
BIOT 474
BIOT 476
BIOT 478
Application of bioinformatics approaches to vitronection
domain identification and functional prediction
Evaluation of clearance of Minute Virus of Mice (MVM) across
an anion exchange column: Influence of viral preparation on
LRV
ROS/pH responsive nanoparticles for therapeutics delivery in
inflammation
Clarification and capture of high concentration refold pools
for E.coli based therapeutics using expanded bed adsorption
chromatography
Jeremy Pike, [email protected], Joanna DeBear, [email protected],
Sankaranarayanan, Jose´ M Morachis, Gloria Kim, Adah Almutairi.
University of California at San Diego, Skaggs School of Pharmacy and
Yuchen Lo1, [email protected], Xiao Meng2, Hiroshi Honda2. (1)
Department of Biomedical Engineering, University of California, Los
Angeles, CA 90095, United States (2) Department of Bioengineering,
Northwestern Polytechnic University, Fremont, CA 94539, United States
Vitronectin has been known as a spreading factor or S factor,
is a multifuction adhesion glycoprotein found in circulation
system that functions in hemostasis and tumor malignancy. The
bioinformatics methods used to investigate the mouse vitronectin,
domain identification using HMM, critical residue prediction,
cellular localization prediction and structural prediction.
BIOT 473
Spatial homogeneity analysis of packed bed chromatography
Andreas Puettmann1, Sebastian Schnittert1, Birgit Stute1, Siarhei
Khirevich2, Ulrich Tallarek2, Eric von Lieres1, e.von.lieres@fz-juelich.
de. (1) Institute of Bio- and Geosciences 1, Research Center Juelich, Juelich,
NRW 52425, Germany (2) Department of Chemistry, Philipps-University
Marburg, Marburg, HE 35032, Germany
Packed bed chromatography is usually modeled in one or two
spatial dimensions, for example by the general rate model. Such
models assume that fluid flow and solute molecule concentrations
are homogeneously distributed over column cross sections.
Moreover, concentration gradients within the beads are at most
considered along the radial coordinate.
These homogeneity assumptions are studied with a threedimensional model of the involved convection, diffusion and
adsorption processes. Due to the complex geometry only bed
sections with up to several hundred spheres can be computed on
personal computers. Simulation studies are performed for random
packings that present sections of small columns with volumes on
the micro-liter scale.
Complex flow profiles and dominant wall effects are observed in
the interstitial volume. The resulting concentration profiles in the
interstitial column volume and within the porous beads can vary
as strongly along the radial coordinate as along the axial coordinate
of the column.
170
Rachael Alford.Purification Development, Alexion Pharmaceuticals,
Cheshire, Connecticut 06410, United States
In this study, we evaluated the impact of viral preparation purity on
the removal of virus from an antibody solution by anion exchange
chromatography. Two grades of viral preparation, one standard
purity and one high purity, were spiked (1% v/v and 5%v/v) into
an antibody solution. The spiked solutions were processed through
an anion exchange column in flow-through mode. Results indicate
that spike purity does have an effect on the final LRV (log reduction
values) obtained. This may allow spiking of higher levels of virus
without significantly affecting column performance and a possible
way to maximize LRV for column steps in purification processes.
Enas A Mahmoud, [email protected], Jagadis
Pharmaceutical Sciences, La Jolla, California 92093-0657, United States
Application of fluorescence spectroscopy toward determination
of controlled release in situ
Conditions in inflamed tissues can be used as a trigger that activates
logic-gate nanoparticles. The nanoparticles are composed of a
polythioether-ketal that is oxidized by reactive oxygen species at its
thioether moieties to form a more hydrophilic polymer that upon
this hydration process can now undergo acid catalyzed degradation
of the polyketal moieties. This allows the particles to stay intact in
normal healthy conditions and only degrade when the two stimuli
occur in tandem as in inflamed tissues. Encapsulated Nile Red
proved the hydrophilicity change upon oxidation as it showed
a hypochromic shift of its fluorescence. The degradation was
confirmed by dynamic light scattering measurements that showed
that the nanoparticles disappeared within 24hrs. Encapsulated
proteins showed complete release only when subjected to low
pH and H2O2. Cell studies showed cytoplasmic release of the
encapsulated protein in macrophages. Future work will apply this
system to diseases characterized by inflammatory areas.
Cathryn L. McFearin, [email protected], Adah Almutairi.School
of Pharmacy and Pharmaceutical Sciences, University of California, San
Diego, La Jolla, California 92093-0600, United States
BIOT 477
BIOT 475
Critical to the development of stimuli-responsive nanomaterial
systems is characterizing their release properties for delivery
of therapeutic or diagnostic agents. Standard methods used to
obtain this information include chromatographic techniques that
require sample degradation and time consuming extraction and
purification before detection. A method capable of probing the
desired release characteristics in situ without having to separate
the payload and delivery system would be advantageous. The work
in this talk shows fluorescence lifetime measurements can achieve
this goal with several advantages over commonly used steady state
fluorescence measurements. This technique is applied to polymeric
nanoparticles using a fluorescent dye that sensitively probes the
complex environment found in delivery systems.
Xuankuo Xu1, [email protected], Jeet Hirapra3, Kevin Epting4,
Siegfried Rieble1, Shih-hsie Pan2, Sanchayita Ghose1. (1) BPPD Process
Development, Downstream, Bristol-Myers Squibb, East Syracuse, NY 13057,
United States (2) BPPD Process Development, Bristol-Myers Squibb, East
Syracuse, NY 13057, United States (3) Rochester Institute Of Technology,
Rochester, NY 14623, United States (4) Purification Technologies, Sartorius
Stedim, Bohemia, NY 11716, United States
Protein refolding at high concentration usually leads to formation
of protein precipitates. Direct filtration of these refold materials
is often very challenging, namely low filter capacity and high
filtration variability. The use of EBA here eliminated the need to
filter the original refold materials. Instead, filtration was performed
only on elution pools with substantially reduced volume (>3fold) and turbidity (>30-fold), hence much easier filtration. The
unique property of the resin used (FastLine SP IEX) allowed EBA
to be operated at very high linear velocity (800-1600 cm/hr),
while still providing sufficient selectivity and impurity clearance.
Furthermore, the relatively broad elution profile of EBA is
particularly suitable for such proteins of low solubility as the one
studied here that readily precipitated in packed bed due to sharp
elution profile. This work demonstrates that the use of EBA helps
debottleneck downstream productivity by achieving efficient
clarification and high-capacity capture in a single step.
Recovery of product from high cell mass cell culture processes
Anne Thomas, [email protected], Tom McNerney, Krista Petty,
Xiaoyang Zhao, Rob Piper.Amgen, United States
Recent advances in upstream produces processes have generated
product titer of in excess of 20g/L. This volumetric productivity
improvement is largely due to significant increase in the cellular
mass. The cellular mass generated throughout the course of
production is between 20- 40% based on packed cell volume (PCV);
well beyond the capabilities of a typical disk stack centrifuge.
In order to harvest product from the high cell mass processes, a
flocculation step was incorporated into the process. Methods for
separating the flocculated cells and debris from the process stream
were evaluated. Three separation methods were assessed for their
feasibility as a potential harvest method for flocculated cell culture
broth; an inclined plate cell settler, an acoustic filter, and alternating
tangential flow micro filtration.
BIOT 479
Synthesis of binding sites in MIP’s system for biosensors
Kyung Choi, [email protected], University of California,
Irvine, California 92697, United States
Recent developments in nanotechnology have brought us new
advances in device fabrications and novel materials by emerging
technologies from physicists, chemists, engineers, biologist, and
materials science. There are a lot of challenges for chemists to play
an important role in this area since nanotechnology is a part of
the chemical domain, which builds up materials at the molecular
level. Microfabrication technology offers us rapid assembly and
integration of unconventional devices since small patterns on a
variety of substrates produced numerous active devices to satisfy
a set of our demands in miniaturization. Microfluidic approach
has taken intensive attractions since microfluidic reactors allow us
to produce novel materials with specific advantages. We present
a microfluidic synthesis of molecularly imprinted polymer
(MIP) with high affinities for biosensors or chemical detection.
171
Wednesday Afternoon
BIOT 472
BIOT 480
SEM comparison of two non-native Tephritid flies of
California: The walnut husk fly and the olive fruit fly
Edmund O Duarte1, Hiroshi Honda2, [email protected],
Wan-Yuan Huang2, Yu-Chen Lo3. (1) Department of Biological Sciences,
California State University, Hayward, CA 94542, United States (2)
Department of Bioengineering, Northwestern Polytechnic University,
Fremont, CA 94539, United States (3) Department of Biomedical
Engineering, University of California, Los Angeles, CA 90095, United States
The fruit flies make good specimens for SEM study. The samples
are rendered with anesthesia, and coated in the sputter coater with
gold/palladium then viewed in the SEM. There are many species of
fruit flies are considered important agricultural pests, and others
are minor or potential pests.
BIOT 481
Development of amperometric biosensor for serum cholesterol
level determination
Edward J Parish1, Yuchen Lo2, Wan-Yuan Huang3, bill720223@
yahoo.com.tw, Hiroshi Honda3. (1) Department of Chemistry and
Biochemistry, Auburn University, Auburn, AL 36849, United States (2)
Department of Biomedical Engineering, University of California, Los
Angeles, CA 90095, United States (3) Department of Bioengineering,
Northwestern Polytechnic University, Fremont, CA 94539, United States
This paper represents the development of a mathematical model
to simulate biosensor kinetic for cholesterol determination. This
model can be validated by comparing its signal output with the
real-time biosensor data to test its accuracy.
Current technologies for converting biomass to fuel products
are severely hindered by cellulose recalcitrance. Recently, it was
found that certain ionic liquids (ILs) can dissolve cellulose. To
understand the molecular origins of cellulose recalcitrance and the
forces provided by ILs in overcoming it, we performed atomistic
molecular dynamics simulations of dissolved and undissolved
cellulose in water and the IL 1-butyl-3-methylimidazolium
chloride (BmimCl). We characterized cellulose’s interaction
network and found that intersheet interactions, specifically
C-H---O contacts, are a primary cause of cellulose recalcitrance.
Comparing the dissolution of cellulose in the two solvents, we
have found that energetically, cellulose dissolution is unfavorable
in water but favorable in BmimCl, and that both the Cl- and
the Bmim+ ions exert disruptive effects on cellulose’s C-H---O
intersheet interactions. Also, the perturbation of solvent structures
by dissolved glucan chains and its effect on solvent entropy can be
a dominant factor in determining cellulose solubility.
BIOT 483
Evaluation of endotoxin removal technologies
Kasey Mackay1, [email protected], Giulia Weissenberger2,
Michael Lihon1, Anne S. London1. (1) Novartis Biologics Center, Novartis
Institutes for Biomedical Research, Cambridge, MA 02139, United States
(2) Department of Biology, State University of New York - Brockport,
Brockport, NY 14420, United States
Endotoxins, small molecules present in bacterial membranes,
pose a risk of contamination in biological products. If not cleared
during the manufacturing process, tissue injury, fever, septic shock
or death can occur if injected into mammals. With both clinical
and commercial manufacturing, steps and technologies must be
developed to clear this impurity from pharmaceutical products.
Here, commercially available technologies are evaluated and
compared for pre-clinical protein production.
Dharmesh Kanani1, [email protected], Navneet
Sidhu1, Elena Komkova1, Amro Ragheb1, Cesar Zuin1, Carl Lawton2,
Amitava Kundu3. (1) Natrix Separations Inc., Canada (2) Massachusetts
Biomanufacturing Center, MA, United States (3) Genmab MN Inc., MN,
United States
Disposable technologies are becoming popular process strategies
particularly in the area of upstream processing in the manufacturing
of biopharmaceuticals. Natrix Separations is developing a
completely disposable hydrogel based chromatographic solution
from capture to polishing steps in the downstream processing of
monoclonal antibodies as well as other biotherapeutics. This study
presents the data on binding capacity, selectivity and throughput
of Natrix hydrogel platform based ion-exchange and hydrophobic
interaction media for the purification of monoclonal antibody
from a representative feed stream.
Maria Williams, [email protected] and
Cellulose recalcitrance, solvation and thermodynamics in
water and in ionic liquids
The basis of my experiment is to the study the reaction of cytochrome
c and cytochrome oxidase using site-directed mutagenesis and
steady-state kinetics. The goal was to characterize the electrostatic
interaction between cytochrome c and cytochrome oxidase. The
mutants that were expressed in this project were E89T/E90Q and
E89T/E90K. These mutants replaced the native negative charge on
glutamic acid 90 [E90] with a neutral charge, glutamine [Q], or
172
Single-use high performance chromatographic media:
Purification of monoclonal antibody in bind and elute mode
Kinetic study of cytochrome C and cytochrome oxidase
Biochemistry, University of Arkansas,
California, Berkeley, Berkeley, California 94720, United States
BIOT 485
BIOT 484
BIOT 482
Adam S. Gross, [email protected], Alexis T. Bell, Jhih-Wei
Chu.Department of Chemical and Biomolecular Engineering, University of
a positive charge, lysine [K]. By gathering data from the kinetic
experiments, we will be able to determine if the mutations had
an effect on the reaction between cytochrome c and cytochrome
oxidase. Steady-state kinetics generally displays saturation kinetics
at sufficiently high cytochrome c concentrations and often obeys
Michaelis-Menten kinetics with a Michaelis constant, Km, and a
maximum velocity, Vmax [2].
Wednesday Afternoon
Molecularly imprinted polymer can be provided by “molecular
imprinting technique”, which is a general protocol for the creation
of synthetic receptor or binding sites with specific molecular
recognition functions in cross-linked network polymers. Synthesis
of high affinity receptor sites is a key contribute to achieve high
sensitivity in their molecular recognition functions.
173
A
B
C
8:30 a.m.
Room# 16A
Downstream Processes: General Topics in Downstream Processing
8:30 a.m Room# 16B
Upstream Processes: Bionanotechnology
S. Wang, N. Varadarjan Papers 494-501
8:30 a.m. Room# 17A
W. Zhou, L. Palomares Papers 502-508
D
8:30 a.m.
Room# 16A
J. Pieracci, S. Hanala, N. Eifler, S. Tobler
Papers 486-493
BIOT 486 – 8:30 a.m.
General Topics in Downstream Processing
J. Pieracci, S. Hanala, N. Eifler, S. Tobler Papers 486-493
Upstream Processes: Cell Culture Development –
Accomplishments and Challenges
Downstream Processes:
8:30 a.m.
Room# 25A Advances in Biotechnology Product Development :
Case Studies for Technology Transfer and Product Commericalization
S. Ozturk, R. Kiss, T. Charlesbois Papers 509-515
Effect of anti-apoptosis genes on clarification performance
Ajish Potty, [email protected], Sonal Patel, Holly Prentice,
Anthony DiLeo, Alex Xenopoulos.Process Solutions, EMD Millipore,
Bedford, MA 01730, United States
Optimal bioreactor harvest time is typically determined based on
maximizing product titer without a negative impact on product
quality. We suggest that ease of downstream purification (DSP)
should also be considered during harvest. In this view, we studied
the effect of anti-apoptosis genes (a cell line modification for
improved cell viability and titer) on downstream performance.
Our hypothesis was that more robust cells would exhibit less cell
lysis and thus generate less cell debris and host-cell contaminants.
We focused on the clarification unit operation, measuring postclarification turbidity and host-cell protein (HCP) concentration
as a function of bioreactor harvest time/cell viability. In order to
mimic primary clarification using disc-stack centrifugation, a scaledown model consisting of a rotating disk (to simulate shear in the
feed zone of the centrifuge) and a swing-bucket lab centrifuge was
used. Our data suggest that in the absence of shear during primary
clarification (typical of depth filters), a 20-50% reduction in HCP
levels was observed for cells with anti-apoptosis genes compared
to control cells without the genes. However, on exposing the cells
to shear levels typical in a disc-stack centrifuge, the reduction in
HCP is only 10-15%. We will discuss the implications of the above
results in detail and suggest possible harvest strategies that may
benefit DSP.
BIOT 487- 8:50 a.m.
Assessment of downstream processing facility bottlenecks due
to cell culture titer improvements
Suma Rao1, [email protected], Oliver Kaltenbrunner1, Sam Guhan2,
Christina Wells3. (1) Purfication Process Development, Amgen, Thousand
Oaks, California, United States (2) Global Process Development, Amgen,
Thousand Oaks, California, United States (3) Global Process Engineering,
Amgen, Colorado, United States
174
Until recently the major bottleneck of monoclonal antibody
manufacturing was considered to be cell culture capacity. In response
to this perceived bottleneck, enormous capital investments were
made throughout the biotech industry to build more cell culture
capacity. At the same time, major efforts to increase cell culture
product titers were undertaken. Recent reported cell culture titers
of 10 g/L and higher highlight the progress made in this area.
However, existing facilities were designed and built assuming
much lower culture titers; this necessitated large reactor volumes
and downstream capabilities sufficient to handle mass output
from these low titer processes. Consequently, when a process with
high cell culture titer is transferred to an existing facility, there
is a discrepancy between upstream and downstream capacity in
this facility. In this study, we will discuss the implications of this
imbalance on facility limitations. We demonstrate that careful
consideration of facility and process capabilities combined with
appropriate modeling will be critical to take full advantage of these
high titer improvements. Otherwise, the impact of the recent major
advances in cell culture technology offer only minor improvements
for process economics of monoclonal antibody processing in
existing facilities.
BIOT 488 – 9:10 a.m.
Affinity chromatographic purification of antibodies by small
peptide ligand from different sources
Zhuo Liu, [email protected], Patrick V Gurgel, Ruben G Cabonell.
Department of Chemical and Biomolecular Engineering, North Carolina
State University, Raleigh, NC 27695, United States
A family of linear hexamer peptide ligands HWRGWV, HYFKFD
and HFRRHL, initially identified for their affinity to the Fc portion
of human immunoglobulin G (hIgG), also have potential for use in
purification of human immunoglobulins A (hIgA) and M (hIgM).
HWRGWV demonstrated the strongest binding affinity to hIgM,
followed by hIgA and hIgG, respectively. Affinity chromatographic
purification of human IgA, IgG and IgM from complete minimum
essential media was achieved at different peptide densities. The
results suggested that the improved recovery at higher peptide
density due to increased binding affinity was compensated by
the decrease in purity for all three immunoglobulins. With these
knowledge, affinity chromatographic purification of human
IgA and IgM from CHO and human B lymphocyte cell lines as
well as one step purification of human IgG, IgA and IgM from
Cohn fraction II/III were investigated. Different pretreatment
methods were employed in order to improve the purity. After
caprylic acid pretreatment or the combination of caprylic acid
and polyethylene glycol pretreatment, highly purified (over 95%
in purity) hIgG was achieved with 60% in overall yield, which is
comparable to chromatographic purification method involving
175
Thursday Morning
Thursday Morning
Sessions
Thursday Morning Sessions
BIOT 489 – 9:30 a.m.
Virus clearance study for RSV F vaccine processing
Deqiang Yu, [email protected], James Leverone, Ashley
Sacramo, Luis Maranga.Technical Development, Novartis Vaccines &
Diagnostics, Cambridge, MA 02139, United States
Respiratory syncytial virus (RSV) is the most important unmet
pediatric vaccine need in developed countries. Our RSV F
subunit vaccine candidate is an engineered recombinant RSV
fusion glycoprotein (F) expressed by CHO-K1 cells. CHO cellexpressed protein has the potential issue of virus contamination
from endogenous retrovirus particles and adventitious agents.
Therefore, virus clearance in RSV F processing is very important
to mitigate the risk of virus contamination. We studied various
virus clearance technologies, including detergent inactivation,
ion exchange chromatography, pH inactivation, membrane
chromatography, and nanofiltration. For different technologies,
RSV F protein stability and recovery were studied and the virus
clearance capability was evaluated by virus spiking study. The virus
clearance steps were chosen and integrated into the RSV F protein
purification process. The total process achieved high log reduction
values for the model viruses and thus mitigated the risk of virus
contamination in this new vaccine candidate.
BIOT 491 – 10:30 a.m.
Detergent-free purification of membrane proteins
Tim R Dafforn1, [email protected], Mohammed Jamshad1,
Yu Pin Lin1, Tim J Knowles3, Mark Wheatley1, David R Poyner4, Roslyn
M Bill4, Rosemary Parslow1, Michael Overduin3, Owen R Thomas2.
(1) School of Biosciences, University of Birmingham, Birmingham, West
Midlands B152TT, United Kingdom (2) Chemical Engineering, University
of Birmingham, Birmingham, West Midlands B1552TT, United Kingdom
(3) Cancer Studies, University of Birmingham, Birmingham, West
Midlands B152TT, United Kingdom (4) School of Life and Health Sciences,
Aston University, Birmingham, West Midlands B4 7ET, United Kingdom
Methods for purifying membrane proteins generally require the
disruption of the membrane using detergents. This removes the
protein from its native lipid environment adversely effecting the
activity of the protein. In this work we show for the first time how
a single low cost reagent, styrene maleic copolymer, can be used to
extract membrane proteins directly from native membranes. The
solubilisation is a rapid single step process that yields a disc-like
nanostructure. This structure is made up of an outer polymeric
annulus enclosing a disc shaped (10 nm diameter) lipid bilayer
which solvates the protein. We show that membrane proteins
solubilised using this method retain native-like activity and are
amenable to conventional downstream processing. We also show
that, where required, styrene maleic acid can be removed and
recycled. This new method opens a whole class of proteins for
future study and application.
BIOT 492 – 10:50 a.m.
BIOT 490 – 10:10 a.m.
Introducing one-step selectivity in the primary recovery of
biotherapeutics
Pim Hermans, [email protected] BV, Leiden, The Netherlands
Affinity chromatography is one of the most effective methods for
purifying protein therapeutics. For standard MAb purification
Protein A is a well established affinity ligand providing the benefits
of a highly selective primary capture step. However, for non
antibody based therapeutics and novel Ab based formats, it becomes
a challenge to find a protein A equivalent. The CaptureSelect
technology, based on camelid single domain antibody fragments,
addresses these challenges and provides a “plug-and-play”
platform approach by introducing a generic capture step for the
primary recovery of virtually any biological product, showing
excellent contaminant clearance and enabling elution conditions
that preserve product structure and activity.
176
Rapid processing of food-derived suspensions using cross-flow
microfiltration for microorganism concentration and recovery
Xuan Li1, [email protected], Eduardo Ximenes1, Hunter Vibbert3,
Xingya Liu4, Kirk Foster2, Michael Ladisch1,2. (1) Department of
Laboratory of Renewable Resources Engineering & Agricultural and
Biological Engineering, Purdue University, West Lafayette, IN 47907,
United States (2) Department of Biomedical Engineering, Purdue
University, West Lafayette, IN 47907, United States (3) Department
of Laboratory of Renewable Resources Engineering & Department of
Chemistry, Purdue University, West Lafayette, IN 47907, United States (4)
Department of Laboratory of Renewable Resources Engineering, Purdue
University, West Lafayette, IN 47907, United States
This paper discusses an automated cross-flow microfiltration
system that concentrates and recovers microorganisms in a timeefficient manner from food-derived suspensions. A hollow fiber
membrane module having a nominal pore size of 0.2 microns
constitutes the core of this cell concentration and recovery (CCR)
system. The system efficiency is investigated using aqueous extracts
of chicken containing Salmonella Enteritidis together with naturally
occurring flora, proteins, lipids, and micron sized particulates.
Both the natural flora in chicken extracts and spiked Salmonella
are concentrated by 500-1000 times within 2 hours with a recovery
of 70% on average, as determined by plating. Buffer composition/
formulation and cleaning are key components in achieving
reproducible trans-membrane fluxes upon multiple uses of the
membrane module. Criteria for membrane chemistry, selection,
and fouling mitigation are presented. The system finds important
application in food borne pathogen detection in response to the
demand for a rapid method of microorganism concentration from
the food matrices.
Upstream Processes:
Bionanotechnology
8:30 a.m Room# 16B
S. Wang, N. Varadarjan Papers 494-501
BIOT 494 – 8:30 a.m.
BIOT 493 – 11:10 a.m.
Molecularly imprinted polymers (MIPs) for selective solid
phase extraction of phospholipids
Robert Sulc, [email protected], Börje Sellergren.Institute
of Environmental Research (INFU) of the faculty of Chemistry, Technical
University of Dortmund, Dortmund, NW 44227, Germany
The purification of pharmaceutical ingredients from endotoxin
impurities is one of the challenges faced by the industry today.
This research focuses on selective solid phase extraction of
phospholipids which stand as surrogates for the lipopolysaccharide
endotoxins. The solid phase consists of molecularly imprinted
polymers (MIPs) which bind the target phospholipid. Cationic
monomers were employed to create the recognition sites, and the
solvent selection was adjusted to optimize the pre-polymerization
complex. The monomer synthesis and polymerization conditions
will be discussed. The free radical polymerization was used to
produce the polymers, which were crushed and sieved to yield
uniform size particles. The subsequent particles were used in
rebinding studies. The produced solids were evaluated for binding
of the target phospholipid, and the results will be discussed.
Enhancing the delivery efficacy of drug-encapsulated
nanoparticles using a transferrin variant
Ricky Y. T. Chiu1, [email protected], Kristine M. Mayle1, Takuma
Tsuji2, Christina T. Liu1, Robert J. Lamm1, Johnny Wang1, Anne B.
Mason3, Daniel T. Kamei1. (1) Department of Bioengineering, University
of California, Los Angeles, Los Angeles, CA 90095, United States (2)
Department of Materials, Physics, and Engineering, Nagoya University,
Nagoya, Japan (3) Department of Biochemistry, University of Vermont
College of Medicine, Burlington, VT 05405, United States
Transferrin (Tf) has been widely studied to target drugs to cancer
cells, since many cancer cells overexpress Tf receptors on their
cell surfaces. However, these approaches have been limited, since
native Tf has a low probability of delivering its payload as it spends
only about 5 minutes inside the cell. To increase the time Tf
spends with a cell, i.e., its cellular association, our group previously
engineered a Tf variant, which was found to exhibit an increase
in cellular association that translated into an improved ability
to deliver a conjugated toxin. In this project, we investigated the
performance of drug-loaded nanoparticles (NPs) decorated with a
Tf variant relative to those conjugated to native Tf. We successfully
demonstrated that the drug-loaded NPs exhibited a higher potency
when conjugated to the Tf variant.
BIOT 495 – 8:50 a.m.
Hyaluronic acid-coated iron oxide nanoparticles for targeted
therapy and diagnosis of cancer and atherosclerosis
Mohammad H El-Dakdouki1, [email protected],
Kheireddine El-Boubbou1, Medha Kamat1, David C. Zhu3,4, George S.
Abela2, Xuefei Huang1,4. (1) Chemistry, Michigan State University, East
Lansing, MI 48824, United States (2) Department of Medicine, Division
of Cardiology, Michigan State University, East Lansing, MI 48824, United
States (3) Departments of Radiology and Psychology, Michigan State
University, East Lansing,, MI 48824, United States (4) Biomedical Imaging
Research Centre, Michigan State University, East Lansing, MI 48824,
United States
177
Thursday Morning
two chromatography steps when proteins are isolated from plasma
fractions. HIgA (40%) and hIgM (45%) enriched immunoglobulins
with over 95% in purity were obtained as the by-products.
BIOT 496 – 9:10 a.m.
Polymeric gold nanorod assemblies for efficient transgene
delivery
Thrimoorthy Potta, [email protected], James Ramos, Kaushal
Rege.Chemical Engineering, Arizona State University, TEMPE, AZ 85281,
United States
Gold nanorods (GNRs) have been explored as promising
nanomaterials for various biomedical applications including
biosensing, imaging, photothermal treatment and drug delivery
for various diseases. In this study, we synthesized a library of
poly(aminoethers)(PAEs) using ring-opening polymerization
reaction between diverse amines and diglycidyl ethers. PAEs
were characterized using 1H NMR, FTIR and GPC analyses. A
library of PAE-GNR assemblies was generated by layer-by-layer
deposition of poly(aminoethers) onto GNRs. The PAE-GNR
assembles exhibited high colloidal stability and DNA binding
capacities due to electrostatic interactions. The role of several
physicochemical properties including, pH-buffering capability,
DNA binding efficacy, polyplex size and zeta potential, of both,
PAEs and PAE-GNR assemblies on transgene expression efficacies
was investigated. Parallel screening of the PAEs and PAE-GNR
assemblies led to identification of several leads that demonstrated
higher transgene expression efficacies and lower cytotoxicities
compared to branched pEI (25 kDa) in different prostate and
pancreatic cancer cell lines. Ongoing work involves investigation
into targeting strategies for selective delivery to specific cell types.
These preliminary results indicate that these assembles have high
potential as theranostics for combined transgene delivery and
imaging.
178
BIOT 497 - Withdrawn
BIOT 498 – 10:10 a.m.
BIOT 499 – 10:30 a.m.
Assembly of photoactive virus capsids on nanoscale templates
Jolene L Lau1, [email protected], Debin Wang1, Stacy Capehart2, Matthew
Cell-targeted photodynamic therapy using virus-like particles
B Francis1,2, James J De Yoreo1. (1) Molecular Foundry, Lawrence
Berkeley National Lab, Berkeley, CA 94720, United States (2) Department
Jin-Kyu Rhee1, [email protected], Michael M Baksh1, Hiroaki
of Chemistry, University of California, Berkeley, CA 94720, United States
Kitagishi1,3, Corwin Nycholat2, James C Paulson2, M.G. Finn1. (1)
Department of Chemistry, The Scripps Research Institute, La Jolla,
California 92037, United States (2) Department of Chemical Physiology
and Molecular Biology, The Scripps Research Institute, La Jolla, California
92037, United States (3) Department of Molecular Chemistry and
Biochemistry, Faculty of Science and Engineering, Doshisha University,
Kyotanabe, Kyoto 610-0321, Japan
Reconstructed viral capsid proteins are attractive nanochemical
scaffolds because of their known icosahedral structures, selfassembled and genetically-controlled protein composition,
robust stability, monodisperse size and shape, biocompatibility,
and polyvalency. They are now commonly used for the display
of multiple copies of cell- and tissue-targeting molecules and
therapeutic agents on their outer surface or interior space. We
describe here the dual functionalization of Qβ virus-like particles
(VLPs) by encapsulation and surface modification technique, in
order to expand the design possibilities of these agents as building
blocks for materials science and medicinal applications.
Qβ VLPs encapsulating multiple copies of fluorescent proteins
were generated in high yields using a modular system enhanced
by specific engineered RNA-protein interactions. The resulting
particles were structurally indistinguishable from recombinant
Qβ alone. The encapsulated proteins were nearly identical
in photochemical properties to monomeric analogues, were
more stable toward thermal degradation, and were protected
from proteolytic cleavage. Residues on the outer capsid surface
were chemically derivatized by acylation and azide-alkyne
cycloaddition without affecting the fluorescence properties of the
packaged proteins. A high affinity carbohydrate-based ligand of
the CD22 receptor was thereby attached, and specific cell labeling
by the particles was successfully detected and quantified by flow
cytometry and fluorescence microscopy. When simultaneously
decorated with metalloporphyrin groups on the exterior surface,
the multifunctional particles showed the ability to kill CD22displaying cells under irradiation by visible light, due to the
porphyrin-mediated generation of singlet oxygen. This type
of system therefore shows promise for targeted photodynamic
therapy, particularly of cells of the immune system.
The bacteriophage MS2 coat protein can be recombinantly
expressed to generate hollow icosahedral capsids stable to a variety
of chemical modifications. Amino acid side chains are displayed
on these monodisperse protein nanoparticles with symmetric,
well-defined spacing. Using unnatural amino acid incorporation
technology, we specifically modified the exterior of MS2
capsids with single-stranded DNA and the interior with organic
fluorophores. Using AFM tip-based fabrication techniques, we
patterned gold surfaces with nanoscale DNA templates to facilitate
capsid assembly. We studied the effects of altering DNA and buffer
composition on 3-D solution assembly and 2-D templated surface
assembly of viral clusters. Encapsulated fluorophores exhibited
energy transfer when capsids were assembled, and we monitored
single-cluster energy transfer with surface confocal microscopy. By
combining top-down, tip-based nanofabrication with bottom-up
assembly of capsids, we can generate novel patterned materials for
molecular detection or artificial photosynthesis.
BIOT 500 – 10:50 a.m.
Cellular and immunological responses toward a polymer
functionalized self-assembling protein nanocapsule
Nicholas M Molino1, [email protected], Kateryna Bilotkach2,
Deborah A Fraser3, Dongmei Ren1, Szu-Wen Wang1. (1) Department
of Chemical Engineering and Materials Science, University of California
Irvine, Irvine, CA 92697, United States (2) Department of Biomedical
Engineering, University of California Irvine, Irvine, CA 92697, United
States (3) Department of Molecular Biology and Biochemistry, University
toward the wild-type and modified nanocapsules by measuring C4
consumption and C5a release. Evidence suggests that conjugation
of PEG to these particles moderately increases the complement
response, relative to E2-WT. Our results demonstrate that
PEGylation of the E2 protein nanocapsules can modulate cellular
uptake and induce complement responses.
BIOT 501 – 11:10 a.m.
Immune responses to chemically attached motifs on virus-like
particles
Marta Comellas-Aragones1, [email protected], Zinaida
Polonskaya1, Lisa Kain2, Shenglou Deng3, Yang Liu3, Paul Savage3, Luc
Teyton2, M.G. Finn1. (1) Department of Chemistry, The Scripps Research
Institute, La Jolla, California 92037, United States (2) Department of
Immunology, The Scripps Research Institute, La Jolla, California 92037,
United States (3) Department of Chemistry, Brigham Young University,
Provo, Utah 84602, United States
Virus-like particles (VLPs) have been shown to be promising vaccine
carriers due to their inherent immunogenicity, a consequence of
their nanometer size, nucleoprotein content, and regular structure.
VLPs possess structures known to atomic resolution and their
surfaces provide multiple functionalities that can be addressed
in a rational manner both chemically and genetically. The
copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction
is a very useful approach for VLP modification, allowing a high
degree of control of the density and spacing of attached antigens.
Glycans, known to have a very low immunogenicity, show an
increased immune response when loaded onto VLPs via CuAAC.
However, immune responses to constituent chemical groups of
the CuAAC, such as alkynes and triazoles are also observed. We
will report on the immunogenicity of added functional groups
during bioconjugation and consideration of unintended immune
responses with respect to therapeutic development.
of California Irvine, Irvine, CA 92697, United States
Self-assembling protein nanocapsules can be engineered for various
bionanotechnology applications. Using the dodecahedral scaffold
of the E2 subunit from pyruvate dehydrogenase, we introduced
non-native surface cysteines for site-directed functionalization. The
modified nanoparticle’s structural, assembly, and thermostability
properties are comparable to the wild-type scaffold (E2-WT),
even after attaching polyethylene glycol (PEG). Cellular uptake
properties of the PEGylated particles were studied, and reduced
uptake by breast cancer and phagocytic cell lines was observed,
relative to E2-WT. Complement activation, an innate immune
mechanism for recognizing and clearing pathogenic material,
was also studied in vitro to determine the complement response
179
Thursday Morning
Developing targeted therapeutic and diagnostic drug
delivery systems will result in the enhanced accumulation of
chemotherapeutic drugs and a built-in contrast agent in the tissue/
organ of interest at levels that permit efficient therapy and sensitive
detection. In this study, we developed hyaluronic acid (HA)-coated
iron oxide nanoparticles and investigated its utility in targeted
cancer chemotherapy and diagnosis, as well as its application
for the early detection of atherosclerotic plaques. We exploited
the interaction between HA and its main receptor, CD44, that is
overexpressed on cancer cells and on atherosclerotic plaques. We
successfully deployed the HA-coated iron oxide nanoparticles as
targeted nanocarriers of doxorubicin for cancer chemotherapy and
as Magnetic Resonance Imaging (MRI) contrast agents for cancer
diagnosis. In addition, two in vivo models (rabbit and mouse) have
been established to assess the utility of the HA-coated iron oxide
nanoparticles as targeted imaging agents for the early diagnosis of
atherosclerosis, thus offering a platform for potential customized
therapy using novel anti-atherosclerotic drugs.
Cell Culture Development –
Accomplishments and Challenges
8:30 a.m. Room# 17A
W. Zhou, L. Palomares Papers 502-508
BIOT 502 – 8:30 a.m.
Understanding transcriptional enhancement in mAb
producing CHO cells
Hussain Dahodwala, [email protected], Susan T Sharfstein,
Sarah E Nicoletti.Nanobioscience, college of nanoscale science and
engineering, Albany, New York 12180, United States
The ever increasing demand for monoclonal antibodies has led to
an interest in understanding productivity rates in CHO cells. To
investigate whether differential transcriptional rates in parental and
progeny cells are the result of altered interactions of transcriptional
machinery with the CMV promoters, well characterized and
commonly occurring transcription factors interacting with CMV
promoter were selected. Using chromatin immunoprecipitation
(ChIP) and electrophoretic mobility shift assays (EMSA), we were
able to quantify the interactions and observe differences in posttranslational modification of transcriptional factors Ap-2 and NfκB.
Our results indicate a difference in binding of these transcriptional
factors to the DNA in higher producer cell lines. Hence, the
enhancement of transcriptional rates may be explained by
improved accessibility of the transgene inserts to the transcriptional
machinery. In most industrial applications, the strong, viral CMV
promoter is used to drive recombinant protein expression. Demethylation of CMV promoters is known to improve accessibility
to transcription. We demonstrated the loss of methylation along
the promoter sequence of transgenes in amplified cell lines using
methylated DNA immunoprecipitation. By carrying out methylated
DNA immunoprecipation and bisulphite sequencing, we were
able to relate the transgene expression to the methylation state of
the promoter region. Based upon our observations, we subjected
the cells to known methyltransferase inhibitors and were able to
increase the productivity of parental cell clones to the same level
achieved by repeated rounds of MTX amplification. Identification
of these factors indicative of higher producers will help develop
selection methods and strategies for cell design that will bring
down costs, reduce timelines for development, and help realize
the conversion of candidate molecules to therapeutics benefitting
patients.
180
BIOT 503 – 8:50 a.m.
Metabolic flux analysis of CHO cells in fed-batch culture
Woo Suk Ahn, Maciek R Antoniewicz, [email protected].
Department of Chemical Engineering, University of Delaware, Newark, DE
scale down runs we confirmed the relation between glucose and
glycation. The close control of glucose concentration in cell culture
through simple approaches can reduce variations in glycation and
acidic variant levels, resulting in more consistent product quality
profiles.
19716, United States
Despite significant investments in research and development
in the past decades, relatively little is known about intracellular
metabolism of CHO cells in cell culture. In this work, the metabolism
of CHO cells was studied in detail during a fed-batch fermentation.
Metabolism was characterized by measured extracellular uptake and
excretion rates of metabolites combined with a detailed metabolic
network model for CHO cells. To obtain additional information
13C-tracers were applied and labeling of intracellular metabolites
was measured using mass spectrometry (GC-MS). Fluxes were
quantified using non-stationary 13C-metabolic flux analysis. The
flux results revealed significant rewiring of intracellular metabolic
fluxes in the transition from growth phase to stationary phase,
including changes in energy metabolism and redox metabolism.
The results that we present provide a solid foundation for future
studies of CHO cell metabolism for applications such as cell line
development and medium optimization for high-titer production
of recombinant proteins.
BIOT 504 – 9:10 a.m.
Achieving consistent and improved product quality profile by
addressing glucose control in a fed batch reactor producing a
recombinant monoclonal antibody
Tom Stapp1, [email protected], Lisa Zheng1, Connie Lu2, Nattu
Vijayasankaran1, Srikanth Chary1, Gargi Seth1. (1) Department of Late
Stage Cell Culture, Genentech, South San Francisco, CA 94080, United
States (2) Department of Protein Analytical Chemistry, Genentech, South
San Francisco, CA 94080, United States
Production of humanized monoclonal antibodies using CHO cells is
a major area of interest in the biotechnology industry. Some product
heterogeneity is introduced in the cell culture unit operation that
cannot be removed in downstream processing. Of interest is the
charge variant level and controlling it within an acceptable range.
There are many sources of charge variants including sialylation,
deamidation, C-terminal lysine cleavage and adduct formation as
in glycation. Glycation is a non-enzymatic process where glucose
binds to a lysine residue, blocking a positive change and increasing
the level of acidic variants. Using data from multiple pilot scale runs
we showed a positive correlation between the level of glycation and
acidic variant level. Taking glucose concentration and product titer
time courses from the runs as input to a kinetic model, we showed
that glucose concentration was a primary cause of glycation. In
BIOT 505 – 9:30 a.m.
Understanding cell nutrient requirements through spent media
analyses and metabolomics: A prerequisite for developing a
high titer cell culture fed-batch process
Ravishankar V Vadali, [email protected], Apara T Oza, Nilesh N Shah,
Pramthesh S Patel.Microbial and Cell Culture Process Development,
GlaxoSmithKline, King of Prussia, PA 19407, United States
The basic principle behind development of a cell culture fedbatch process is to increase the area under the cell growth curve
(IVC) while maintaining the specific productivity of the cells to
maximize the final product yield. During process development of a
mAb product, the area under the cell growth curve was increased
significantly through feed development and process optimization.
However, the final product yield increased only marginally due to
a loss in specific productivity of the cells. Spent media analyses
revealed that three key amino acids were limiting in this cell culture
fed-batch process. The feeds designed for increasing IVC were
then fortified with these specific amino acids to restore the specific
productivity of the cells. Furthermore, a detailed metabolomic
study was performed to analyze the intracellular metabolites
of these cells and extracellular nutrient environment. The data
indicated that, as the culture progressed, the cells encountered
limitations in the following metabolic pathways - (1) Amino
acid biosynthesis, (2) Vitamin biosynthesis, (3) Phospholipid
metabolism, and (4) Nucleotide and Nucleoside metabolism.
Interestingly, limitation in any of these metabolic pathways did not
affect the growth profile of the cells but exhibited a negative impact
on productivity. The media and feeds were augmented with specific
nutrient groups tailored to mitigate the corresponding limitations
with an objective of improving the process performance. The
final optimized fed-batch process delivered multi-gram per liter
of titer. This experience demonstrates the utility of spent media
and metabolomic analyses to understand cell line specific nutrient
requirements while optimizing a cell culture fed-batch process.
BIOT 506 – 10:10 a.m.
Biomanufacturing operations: Chapter and verse
Michael Kamarck, [email protected], United
States
Less than forty-years ago it was the vision of early biotechnologists,
dominated by classically trained chemical engineers, biologists,
chemists, physiologists, and medical professionals, as to whether
recombinant DNA approaches could be exploited for production
and commercialization of therapeutic proteins – a new class of
biological entities targeting novel mechanisms for our most elusive
unmet medical needs. Today, that vision has not only become a
reality, but it has been extended by bioprocess development groups
enabling a broad range of biological motifs, covering numerous
disease targets, and production scales to not only be demonstrated
and manufactured successfully, but for the first time, enabling
access to under-served markets and populations. As we confront
the vibrant future of biologics and vaccines, where it is estimated
that novel entities and biosimilars will constitute significant major
advances in oncology, cardiovascular disease, infectious disease,
and novel mechanisms, bioprocess development will once again be
required and paramount.
BIOT 507 – 10:50 a.m.
Chemically defined media development improves signal
peptide processing: A case study at the 2kL scale
Arvia E. Morris, [email protected], Rebecca McCoy.Cell Sciences
and Technology, Amgen, Seattle, Washington 98119, United States
In recent years Amgen, has incorporated chemically defined
production media and feeds into their mammalian platform
for recombinant protein expression. In this study, the impact on
expression and product quality was examined at the 2kL scale for
a monoclonal antibody. Results are shown for three chemically
defined production and feed media, each from a different stage of
a performance improvement development cycle. The study found
that reformulated production media and feeds improved specific
productivity, titer, and processing of the signal peptide for the
heavy chain of the antibody product. The final reformulated media
has essentially the same ingredients as the original formulation,
but the amount of different ingredients in the culture at a given
time has significant impact on product quantity and quality.
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Thursday Morning
Upstream Processes:
Rapid, large-scale manufacture of immunotherapeutics
Chris M Warner, [email protected], Matthew S Croughan.Amgen
Bioprocessing Center, Keck Gradutate Institute, Claremont, CA 91711,
United States
Manufacture of immunotherapeuctics (MABs) is of great interest
for many medical and industrial applications. Many cell culture
processes exist for these purposes yet suffer from either nonhuman-like glycosylation or slow development times. We propose
to use large-scale, transient transfection of CHO cells as a superior
approach for very rapid production of antibodies with human-like
glycosylation and full effector function. In order to increase titer
and achieve large scale production, we have investigated various
scaling factors of the key physio-chemical parameters involved
in the transfection process through scale down experiments.
These parameters include cell concentration, PEI/DNA complex
concentration, medium formulation, mixing time, and incubation
temperature and time, as well as agitation power per unit volume
under various geometries. Fundamental engineering models,
including Sherwood number analyses of mass transfer rates, are
used to explain the results and predict performance upon scale up
to 25,000 liter stirred tank bioreactors.
Advances in Biotechnology
Product Development :
Case Studies for Technology Transfer and
Product Commericalization
8:30 a.m.
Room# 25A S. Ozturk, R. Kiss, T. Charlesbois Papers 509-515
to seed expansion, where cell growth challenges led to real time
manufacturing investigations. Investigational results suggest that
tight parameters maybe meaningless for seed expansion. With the
possibility of challenges in seed expansion, it is critical to know
which parameters are actually important as you move your product
into commercialization.
BIOT 510 – 8:50 a.m.
Process scale up and tech transfer of new ERT biologic:
Efficient balancing act of people, process, and plants yields
optimized large scale process
Jonathan Blackie, [email protected] Manufacturing,
BioMarin Pharmaceutical Inc., Novato, CA 94949, United States
Biomarin develops and commercializes enzyme replacement
therapies (ERT) to treat different forms of Mucopolysaccharidoses
(MPS). The process to manufacture these products has rapidly
moved from Process Development, to pilot-scale clinical
manufacture, and to commercial-scale manufacture. The process
uses long-term, high cell density perfusion cell culture and has
been optimized using highly flexible and disposable manufacturing
systems. In this talk we will describe the history, process and
approach to tech transfer of ERT products as they move through
the different groups, stages and facilities as well as the technical
challenges, validation strategy and product comparability at scale
up and transfer.
BIOT 511 – Withdrawn
BIOT 512 – 9:30 a.m.
Challenges associated with the technology transfer of mature
processes
Jason Towart, [email protected], South San Francisco, CA
94080, United States
BIOT 509 – 8:30 a.m.
What is the objective of seed expansion and which parameters
are useful
Douglas H Osborne1, [email protected], WeiWei
Hu1, Erik H Hughes2, Thomas Ryll1. (1) Department of Cell Culture
Development, Biogen Idec, Research Triangle Park, North Carolina 27560,
United States (2) Department of Manufacturing Sciences, Biogen Idec,
Research Triangle Park, North Carolina 27560, United States
The manufacturing process for a therapeutic protein can be
remarkably different from a platform monoclonal antibody
process. The introduction of these manufacturing processes into
a new facility can be a challenging endeavor regardless of the
manufacturing process. Case studies comparing and contrasting
the unique challenges encountered during the tech transfers of a
therapeutic protein and an antibody process will be discussed.
BIOT 513 – 10:10 a.m.
BIOT 515 – 11:10 a.m.
Applying QbD concept to biologics drug substance site transfer
Prevnar-13 launch and production network: How many
transfers for one product?
Harry Lam, [email protected] Manufacturing Science and
Technology, Genentech, South San Francisco, CA 94080, United States
Quality by Design (QbD), as articulated in ICH Q8, Q9 and Q10
guidance documents, enables enhanced process understanding and
a more systematic and scientific approach to development. The end
goal is more robust manufacturing processes with better controls
than those that typically result from traditional approaches to drug
development.
The QbD framework has many implications for manufacturers and
regulators alike. This presentation describes how QbD concepts
and risk-based approaches can be applied to improve transfer of
biologics manufacturing processes between sites. It also describes
a novel approach to develop a regulatory strategy for site transfers
that leverages Genentech’s extensive site transfer history, product
and process knowledge, Quality Risk Management and Quality
Systems to allow for QbD post-approval changes including site
transfers.
BIOT 514 – 10:50 a.m.
Daniel R. Lasko, [email protected], Mireli Fino, Willard F.
Waterfield.Bio-Manufacturing Sciences Group, Pfizer, Inc., United States
Prevnar-13 is a multi-serotype conjugate vaccine directed against
pneumococcal disease. The vaccine is one of the most complex
biological products ever approved and has a complex multi-site
manufacturing supply chain. The polysaccharide antigens are
extracted and purified from each individual microbial serotype
following fermentation. The individual serotype-specific purified
polysaccharides are then conjugated to a purified carrier protein in
a series of chemical reaction and purification steps carried out at a
separate location from the production of the polysaccharides. The
final stages of manufacture of the vaccine drug product involves
formulation and aseptic filling into vials and syringes.
This presentation will address the challenges involved in bringing
this highly successful, high volume biotech product to the market
through the lens of the tech transfer process teams charged with
implementing a commercial supply platform from this multimode supply chain.
Assessing and mitigating technology transfer risks
Sourav Kundu, [email protected] & Product Engineering,
Amgen, Inc., West Greenwich, RI 02817, United States
Technology transfer is an integral part of biotechnology product
commercialization lifecycle. Scale-up and technology transfers
often pose high risk to product comparability for complex biological
processes if the process is susceptible to scale and equipment
differences. We recently conducted successful scale-up and
technology transfer of one licensed drug and another in Phase 2/3
development. A thorough risk assessment considering all aspects
drug manufacturing was conducted including the differences in
scale, equipment, raw materials, assays/instrumentation, operating
practices, and process/material flow. The risks were rated for severity,
occurrence and detection based on development/characterization
information, product knowledge, manufacturing history and
technical understanding. High-ranking items were mitigated to
reduce overall risk by either minimizing the number of changes, or
by performing additional laboratory studies (impacting severity),
improving procedures and training (improving occurrence), and
implementing tools and technologies proactively to improve
detection. All comparability criteria were met during subsequent
campaigns demonstrating success of the strategy.
Two technology transfer case studies will be examined related
182
183
Thursday Morning
BIOT 508 – 11:10 a.m.
Upstream Processes:
Bionanotechnology
2:00 p.m Room# 16B
N. Varadarajan, S. Wang Papers 516-523
B
C
2:00 p.m Room# 16B
Upstream Processes: Bionanotechnology
N. Varadarajan, S. Wang Papers 516-523
D
Development of an engineered beta roll motif for the creation
of stimulus-responsive proteinaceous hydrogels
Kevin Dooley1, [email protected], Raymond Tu2, Scott Banta1.
BIOT 516 – 2:00 p.m.
2:00 p.m. Room# 17A
Upstream Processes: Cell Culture Process Development –
Accomplishments and Challenges
L. Palomares, W. Zhou Papers 524-531
2:00 p.m. Room# 25A
Advances in Biotechnology Product Development:
Innovation in Process Development and Manufacturing
D. Roush, J. Coffman Papers 532-538
(1) Department of Chemical Engineering, Columbia University, New York,
New York 10027, United States (2) Department of Chemical Engineering,
The City College of New York, New York, New York 10031, United States
Stimulus-responsive hydrogels are being increasingly developed
for applications including drug delivery, tissue engineering and
biosensors. We have developed a peptide that forms a calciumdependent hydrogel using a rationally engineered beta roll peptide.
In the absence of calcium, the peptide is intrinsically disordered.
Upon addition of calcium, the peptide forms a corkscrew-like
structure. We have mutated one face of the beta roll to contain
leucines, so that structural formation will enable dimerization.
We have added a leucine zipper forming helical domain to the
engineered beta roll and we have shown that these constructs
are able to form hydrogels by physical crosslinking in calcium
rich environments. The structural properties of the mutant beta
roll have been compared to the wild type by circular dichroism,
bis-ANS binding and terbium binding. The elastic and viscous
moduli of the hydrogels have been measured at various calcium
concentrations using a particle tracking technique.
BIOT 517 – 2:20 p.m.
Microenvironmental modulation of cancer stem cells through
engineered bioscaffold
Sharmistha Saha1, [email protected], Pang-Kuo Lo2, Xinrui
Duan1, Hexin Chen2, Qian Wang1. (1) Department of Chemistry and
Biochemistry &Nanocenter, University of South Carolina, Columbia,
Columbia, SC 29208, United States (2) Department of Biological Sciences,
University of South Carolina, Columbia, Columbia, SC 29205, United
States
A large number of literature studies reveal that microenvironmental
interactions control fate of cancer stem cells, like self renewal,
differentiation, invasion, and distant metastasis. Tumor cells
communicate with the surrounding environment, sending and
184
receiving topographical and molecular cues that direct various
cellular phenomena. However, the complexity of the invivo system
makes it difficult to isolate and study those ECM topographical
cues which affect such cellular transitions and behaviors. Thus it
is necessary to employ an invitro biomimetic scaffold which can
simultaneously recreate the natural instructive microenvironments
in terms of matrix composition, topography and spatial orientation
to investigate such cell-matrix interactions. We have previously
established that polymeric electrospun scaffold can induce EMTlike changes in cancer cells by a phenotypic variation through
contact guidance. Our current work emphasizes composite
biological cues from ECM proteins like collagen, fibronectin and
laminin, and geometric cues from electrospun 3D scaffold in
determining cancer stem cell fate. The results of this study indicate
that the microenvironmental cues may play a significant role in
tumor progression and development and can be a novel target for
designing cancer treatment.
BIOT 518 – 2:40 p.m.
siRNA transport and delivery: A time series comparison
between 2- and 3-dimensional cell culture in collagen matrices
Amanda P. Malefyt1, [email protected], Elizabeth Hinds1, Arul
Jayaraman3, Catherine Kuo4, Kyongbum Lee5, Christina Chan1,2, S.
Patrick Walton1. (1) Department of Chemical Engineering and Materials
Science, Michigan State University, East Lansing, Michigan, United States
(2) Department of Biochemistry and Molecular Biology, Michigan State
University, United States (3) Department of Chemical Engineering, Texas
A&M University, United States (4) Department of Biomedical Engineering,
Tufts University, United States (5) Department of Chemical and Biological
Engineering, Tufts University, United States
Difficulty in developing short, interfering RNA (siRNA)
therapeutics for systemic in vivo delivery can be partially attributed
to the inability to deliver effective concentrations of siRNAs to the
cells of interest. While laboratory research studies involving nucleic
acid therapies are typically performed on two-dimensional (2D)
cell cultures, this is not an ideal model of the three-dimensional
(3D) in vivo environment. It has been shown that cells function
differently when cultured within a 3-dimensional matrix. As such,
we are seeking to optimize transfection of siRNAs in 3D, specifically
to cells cultured within collagen gels. Commercial transfection
reagents with proven efficacy in 2D siRNA transfections have
difficulty silencing cells suspended within hydrogel matrices,
primarily due to interactions between the transfection complexes
and the matrix.
In this study, we used multicolor fluorescence confocal microscopy
and flow cytometry to analyze the cellular uptake and efficiencies
of siRNAs delivered through 3D collagen matrices at various time
points (8 to 72h). Delivery was compared using Lipofectamine
2000 (LF2K) and 3-D FectIN, a transfection reagent developed
185
Thursday Afternoon
Thursday
Afternoon
Thursday Afternoon Sessions
BIOT 519 – 3:00 p.m.
Toward backpacking bacteria for diagnostics and therapeutics
Rohan Fernandes1, David Gracias1,2, [email protected]. (1)
Department of Chemical and Biomolecular Engineering, Johns Hopkins
University, Baltimore, Maryland 21218, United States (2) Department of
Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, United
States
We present “backpacking bacteria” – biohybrid devices comprised
of bacteria attached to micro/nano scale cargo. Backpacking
bacteria combine the advantages of bacteria and cargo for use in
diagnostic and therapeutic applications. Bacteria offer numerous
advantages on account of their sizes, their ability to respond to
diverse stimuli, to convert chemical energy into motion and to
grow naturally in niches within the body. Recent advances in
micro/nanotechnologies have enabled the fabrication of micro/
nano scale cargo of controlled sizes, shapes, geometries with
tunable properties such as optical, electrical or magnetic properties.
In our work, we investigate mechanisms of conjugating bacteria
to cargo via non-specific, charge or antibody based interactions.
Additionally, we vary the size, shape and material of the cargo
conjugated to the bacteria. We investigate the properties of the
resultant biohybrid such as the motility/chemotactic response of
bacteria and magnetic/optical properties of the cargo. Prospects
of utilizing backpacking bacteria for extra and/or intracellular
delivery of diagnostic or therapeutic cargo are envisioned.
BIOT 520 – 3:40 p.m.
Targeting and activating antibodies were not essential for
activation of dendritic cells by ovalbumin encapsulated in pH
sensitive hydrogel microparticles
Enas A Mahmoud1, [email protected], Laura E Ruff2, José M
Morachis1, Carol D Katayama3, Maripat Corr4, Stephen M Hedrick3,
Adah Almutairi1. (1) University of California San Diego, Skaggs School of
Pharmacy & Pharmaceutical Sciences, La Jolla, CA 92093-0657, United
States (2) University of California San Diego, Biomedical Sciences, La Jolla,
186
CA 92093, United States (3) University of California San Diego, Division
of Biological Sciences, La Jolla, CA 92093, United States (4) University of
BIOT 522 – 4:20 p.m.
California San Diego, School of Medicine, La Jolla, CA 92093, United States
Single-cell manipulation using nanopipettes
pH sensitive hydrogel microparticles have shown potential as an
antigen carrier and further investigation on the effect of targeting
and activating antibodies on the surface of particles was needed.
Herein, we report microparticles encapsulating ovalbumin at
40% efficiency and decorated with DEC205, CD40 and/or HA
antibodies. The activity of the HA antibody was confirmed by
measuring its interaction with the corresponding peptide. Cell
uptake studies showed uptake and presentation of ovalbumin
by BMDC in-vitro as well as dendritic cells and monocytes invivo irrespective of the type of antibody on the particle surface.
Further investigation of CD80 and CD86 upregulation didn’t show
the effect of antibodies in both in-vitro and in-vivo. Further invivo ovalbumin-expressing vesicular stomatitis virus (VSV-OVA)
challenge studies showed expansion of cytokine-producing CD8
T cells using two different strategies, accelerated vaccination using
pre-loaded BMDC and a traditional mouse immunization setting.
Paolo Actis, [email protected], Boaz Vilozny, Nader Pourmand.
Department of Biomolecular Engineering, UC Santa Cruz, Santa Cruz,
California 95064, United States
BIOT 521 – 4:00 p.m.
To fully understand cell functioning and stochastic gene expression
patterns, it is necessary to measure molecular interactions at
the single-cell level. Nanotechnology-based tools having high
sensitivity and low invasiveness hold great promise as new
biomedical devices for single-cell manipulation. Unlike other
platforms, nanopipettes can be integrated with nanomanipulators,
allowing precise positioning with nanometer resolution. The fully
electrical read-out as well as the ease and low cost of fabrication are
unique features that give this technology enormous potential. The
electrically controlled positioning of the needle-like nanopipette
enables the nondestructive injection, aspiration, and manipulation
of individual living cells. We will present preliminary data showing
that electro-wetting allows the aspiration of minute amount of
cytoplasmic material from individual cells without comprising cell
viability.
Bioorthogonal nanosensors for magnetic and optical detection
of infectious pathogens
BIOT 523 – 4:40 p.m.
Hyun Jung Chung1, [email protected], Thomas
Microfluidic retroreflector diagnostics
Reiner1, Ghyslain Budin1, Changwook Min1, Monty Liong1, David
Issadore1, Hakho Lee1, Ralph Weissleder1,2. (1) Center for Systems
Biology, Massachusetts General Hospital, Boston, MA 02114, United States
(2) Department of Systems Biology, Harvard Medical School, Boston, MA
02115, United States
The ability to rapidly diagnose infectious pathogens would have
far reaching biomedical and technological applications. Here
we describe the bioorthogonal modification of small molecule
antibiotics (vancomycin and daptomycin), which bind to the cell
wall of gram-positive bacteria. The bound antibiotics conjugates
can be reacted orthogonally with tetrazine-modified nanoparticles,
via an almost instantaneous cycloaddition, which subsequently
renders the bacteria detectable by optical or magnetic sensing.
We show that this approach is specific, selective, fast and
biocompatible. Furthermore, it can be adapted to the detection of
intracellular pathogens. Importantly, this strategy enables detection
of entire classes of bacteria, a feat that is difficult to achieve using
current antibody approaches. Compared to covalent nanoparticle
conjugates, our bioorthogonal method demonstrated 1-2 orders of
magnitude greater sensitivity. This bioorthogonal labeling method
could ultimately be applied to a variety of other small molecules
with specificity for infectious pathogens, enabling their detection
and diagnosis.
signal intensity from each 1 mm2 array of retroreflectors. The assay
is implemented in a microfluidic format to enhance reproducibility,
using Rickettsia conorii as a model analyte. The magnetic properties
of the bead labels are useful in sample preparation from complex
matrices and pre-concentration to increase sensitivity, while fluidic
force discrimination and PEGylation of beads and sensing surfaces
are used to increase specificity.
Upstream Processes:
Cell Culture Process Development –
Accomplishments and Challenges
2:00 p.m. Room# 17A
L. Palomares, W. Zhou Papers 524-531
BIOT 524 – 2:00 p.m.
Balakrishnan Raja1, [email protected], Jennifer
D Knoop1, Eliedonna Cacao1, Tim Sherlock2, Archana Kar1, Steven
Kemper1, Gavin Garvey1, Katerina Kourentzi1, Paul Ruchhoeft2, Juan
Olano3, Robert Atmar4, Ronald Renzi6, Anson Hatch5, Richard Willson1.
(1) Department of Chemical and Biomolecular Engineering, University of
Houston, Houston, Texas 77004, United States (2) Department of Electrical
and Computer Engineering, University of Houston, Houston, Texas
77004, United States (3) Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555, United States (4) Department of
Medicine, Baylor College of Medicine, Houston, Texas 77030, United States
(5) Department of Biotechnology and Bioengineering, Sandia National
Laboratories, Livermore, California 94551, United States (6) Microfluidics
Research Group, Sandia National Laboratories, Livermore, California
94551, United States
Retroreflectors return light directly to its source and are easily
detectable using inexpensive optics. This work introduces
planarized, microfabricated linear retroreflectors as bio-sensing
surfaces, using micron-sized magnetic beads as light-blocking
labels in a semi-homogeneous format, resulting in a highly
sensitive diagnostic immunoassay. The target is sandwiched
between magnetic beads and the sensing surface, both decorated
with anti-target antibodies, thereby dimming the retroreflectors. A
highly sensitive and fully automated difference imaging algorithm
that can see single 1.0 μm particles is used to detect and quantify
Molecular mechanism of antibody disulfide bond reduction in
CHO cell culture processes
Kristen Koterbra, [email protected], United States
During large-scale production of a monoclonal antibody product,
we observed significant reduction of the antibody’s interchain
disulfide bonds. This reduction event culminated in lost product as
the bulk failed to meet product quality specifications. Small-scale
laboratory studies revealed that the cause of the reduction was due
to mechanical shearing of the cells, which resulted in the release
of cellular enzymes that in turn reduced the antibody product.
To further investigate the mechanism, the cytosolic isoform of
thioredoxin, TXN1, was knocked down by lentiviral-mediated
RNAi to determine if inhibiting its expression and/or activity could
attenuate (or prevent) antibody disulfide reduction. The results
of these experiments provided the rationale to design a strategy
to develop a host cell line that is devoid of antibody disulfide
reduction and to further analyze how the thioredoxin pathway may
be modulated during our CHO cell culture processes.
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Thursday Afternoon
specifically for hydrogel delivery. Although our results showed
equivalent siRNA uptake under multiple conditions at longer
timepoints (e.g. 48h), effective silencing has only been achieved
through using 3-D FectIN for delivery during gel formation.
Additionally, treatments after gel formation demonstrate significant
siRNA accumulation at the top of the matrix with minimal
penetration throughout the gel. We will discuss how differences in
siRNA accumulation at earlier time points may affect the potential
for silencing at later time points as well as other matrix formulation
methods to aid in delivery throughout the collagen scaffolds.
BIOT 527 – 3:00 p.m.
Non-invasive monitoring of dissolved oxygen and carbon
dioxide in upstream bioprocess development
Dynamic transcriptome profiling for fed-batch and perfusion
bioreactor process characterization
Priyanka A Gupta, [email protected], Jose R Vallejos, Yordan V.
Karthik P Jayapal, [email protected], Chetan T Goudar.
Kostov, Xudong Ge, Govind Rao.Department of Chemical, Biochemical and
Environmental Engineering, University of Maryland Baltimore County,
Baltimore, MD 21250, United States
Cell culture techniques are carried out in small scale disposable
vessels (volumes 0.1 ml to 100ml) in both industry and academia.
Despite the widespread use of these vessels, important parameters
like dissolved oxygen and carbon dioxide concentrations are usually
not monitored.In the past few years there has been significant
development in the field of optical fluorescence sensor technology
and low cost, non-invasive, disposable sensors for dissolved
oxygen and carbon dioxide have become available. These consist
of a fluorescent dye physically immobilized in a gas permeable
matrix. However the possibility of dye leaching and sterilization
issues prevent their facile adoption by the scientific community.
Here a simple and unique solution to this problem is proposed.
We take advantage of analyte (CO2 and O2) diffusion through the
walls of disposable vessels. The sensing patch is placed in a cavity
milled inside the vessel wall and is sealed to prevent interference
from outside atmosphere. The reduced thickness of the vessel wall
due to milling allows diffusion of analyte which is then detected
by the sensor, making online monitoring possible. This process
does not involve physical opening of the vessel and sensor does not
contact the cell culture so the sterility of vessel is not compromised
in any way.
Cell Culture Development, Bayer HealthCare, Berkeley, CA 94710, United
States
The importance of mammalian cell culture processes in recombinant
biotherapeutic proteins manufacturing is unquestionable. Yet, very
little is known about the dynamics of these processes at a molecular
level. Most of the existing literature on this subject pertains to nonproducing cells or those in exponential growth phase which may
not be directly relevant in commercial settings. In this study, we
employed DNA microarrays to analyze the temporal transcriptome
profiles of commercial recombinant protein-producing cells in
two processes – (1) a ~2 week mAb fed-batch process and (2) a
long-term high-flow non-mAb perfusion process. In the case
of the fed-batch process, the cellular transcriptome dynamics
reflects on the non-steady state nature of the process and resultant
adaptation of cells to the changing external environment. Clusters
of gene expression profiles that correlate with growth, viability
and productivity changes were identified. In the case of perfusion
process, despite the presumable steady-state nature of the process,
we identified certain subtle changes in cell’s transcriptome which
correlated with cell age and other important process variables.
In addition to the molecular insights gained from such analyses,
we also demonstrate the utility of such tools in comprehensive
molecular level process comparisons – in this particular example,
for validation of scale-down process models.
BIOT 526 – 2:40 p.m.
BIOT 528 – 3:40 p.m.
2D HPLC-based process analytical technology for bioreactor
control
Controlling trisulfide modification in recombinant monoclonal
antibody produced in cell culture
Erik K Read, [email protected], Kurt A Brorson.Office of
Biotechnology Products, CDER/FDA, United States Food & Drug
Administration, Silver Spring, MD 20903, United States
Alan Gilbert, [email protected], Rashmi Kshirsagar, Kyle
McElearney, Marty Sinacore, Thomas Ryll.Cell Culture Development,
Biogen Idec, Cambridge, MA 02142, United States
One of the goals of the FDA Process Analytical Technology
(PAT) framework is to “enhance understanding and control the
manufacturing process”. To this end, we have developed a flexible
two dimensional high performance liquid chromatography
design for automated, near-real time, at-line bioreactor process
monitoring of product quality attributes from in-process materials.
Application of this technology to a bench scale mammalian cell
culture bioreactor will be described and opportunities for attributebased (e.g. titer, aggregation, glycans) PAT, as well as limitations,
will be discussed.
Molecular heterogeneity was detected in a recombinant
immunoglobulin (IgG1) antibody and attributed to the presence
of a protein trisulfide moiety. Trisulfide bond formation, of which
the predominant modification site was the bond between the heavy
and light chains, has been recently observed in IgGs produced in
cell culture. To minimize heterogeneity and control product quality,
an understanding of the impact of cell culture process conditions
on trisulfide formation is desirable. Cell culture parameters were
varied in bench scale bioreactor studies to investigate the impact on
trisulfide formation. Trisulfide analysis of the samples from these
188
experiments revealed that the trisulfide content varied considerably
from <1% to 39%, despite similar growth and productivity. In
particular, culture duration and feeding strategy were important
variables. As a result of tightly controlling cell culture conditions,
antibody with reproducible trisulfide levels can be produced.
Strategies for controlling trisulfide levels in cell culture will also
be discussed.
BIOT 529 – 4:00 p.m.
Toward online control of glycosylation in MAbs
Melissa M St. Amand, [email protected], Anne S Robinson,
Babatunde A Ogunnaike.Chemical Engineering, University of Delaware,
Newark, Delaware 19716, United States
Glycosylation, a post-translational modification in which a
carbohydrate chain is added to a protein, is an important quality
attribrute affecting monoclonal antibodies (MAbs). MAbs
validated for use as human therapeutics have precise glycosylation
patterns that must be accurately replicated for the MAb to function
as intended in vivo. However, glycan formation and attachment are
subject to variability and are often non-uniform. Consequently,
regulatory agencies are now encouraging biopharmaceutical
manufacturers to develop strategies to control glycosylation online
during production. However, online glycosylation control is yet to
be implemented in the biopharmaceutical industry.
Our goal is to develop—and validate experimentally—a
comprehensive strategy for effective real-time, on-line control
of glycosylation patterns, using a combination of multi-scale
modeling, hierarchical control, and state estimation. In this
presentation, we will discuss work completed thus far to achieve
this goal. First we will discuss a method for determining the
controllability of glycosylation and demonstrate how this
method, which is predicated upon employing statistical design
of experiments to carry out glycosylation model simulations
systematically in order to obtain a glycosylation process gain matrix,
was used to determine the conditions under which glycosoylation
is controllable. We will then discuss the development of a novel
bioreactor system equipped with an OPC interface that has made
possible the implementation of on-line feedback control of glucose
and glutamine concentrations, both key process variables affecting
glycoyslation. The challenges associated with nutrient control
have been systematically addressed through the implementation
of a PID controller with data filtering and gain scheduling.
Experimental results demonstrating the bioreactor system’s ability
to maintain desired nutrient set-points with this control strategy
will be presented. In addition, progress to date on the development
of an at-line assay for glycosylation macro-heterogeneity will be
presented along with preliminary results from a simple proof-ofconcept glycosylation controller.
BIOT 530 – 4:20 p.m.
Improvements in mammalian cell culture by use of metabolic
flux analysis
Nitin Agarwal, [email protected], Robert H Walters,
Jeffrey P Thompson.Process Cell Culture / Fermentation, MedImmune,
Gaithersburg, MD 20878, United States
Empirical approaches for media development in the biotech
industry, while effective are limited to the discovery of local optima
in media composition. In comparison, constraint-based analysis
of metabolic networks offers a facile and rational approach for
global optimization of cell culture media as demonstrated in this
study. We initially developed a metabolic model consisting of 46
metabolites and 78 fluxes, which primarily represented glycolysis,
amino-acid metabolism, TCA cycle, biomass and antibody
production. A linear programming-based method was then used
to optimize for production of cellular biomass. Based on the model
predictions for amino-acid consumption, the relative levels of the
various amino acids were adjusted to create a novel feed medium
that yielded a 40% increase in the total viable cell-count by day 6
of fed-batch cell culture. As a continuation of this effort, a larger
model was subsequently created that included additional pathways
such as lipid, cholesterol and nucleotide metabolism.
BIOT 531 – 4:40 p.m.
Kinetic study of endogenous unfolded protein response and its
applications in CHO production culture
Zhimei Du, [email protected], Dave Treiber, Becca McCoy, Pranhitha
Reddy.Cell Science & Technology, Amgen Inc, Seattle, Washington 98119,
United States
Unfolded protein response (UPR) is the primary signaling
network activated in response to the accumulation of unfolded
and/or misfolded protein in the endoplasmic reticulum (ER). The
expression of high levels of recombinant proteins in mammalian
cell cultures have been linked to increased UPR. However, the
kinetics of different UPR –mediated events and their impact
on cell performance and recombinant protein secretion during
production are ill defined. We created an UPR-responsive,
fluorescence-based reporter system to detect and quantify specific
UPR-mediated transcriptional activation of different intracellular
signaling pathways. We generated stable antibody-expressing
clones containing this UPR responsive system and established
FACS-based methods for continuous, real-time monitoring of
endogenous UPR activation in cell cultures. We found that clones
differed in their UPR induction pattern; both the timing and the
degree of UPR-induced transcriptional activation were linked to
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Thursday Afternoon
BIOT 525 – 2:20 p.m.
Advances in Biotechnology
Product Development: Innovation in
Process Development and Manufacturing
2:00 p.m. Room# 25A
D. Roush, J. Coffman Papers 532-538
BIOT 532 – 2:00 p.m.
Reducing clinical manufacturing and development costs: is
continuous chromatography the answer?
James Pollock1, [email protected], Glen Bolton2, Sa V Ho2,
Daniel G Bracewell1, Suzanne S Farid1. (1) Department of Biochemical
Engineering, Unversity College London, London, London WC1E 7JE,
United Kingdom (2) Pfizer R&D Global Biologics, Andover, MA 01810,
United States
During clinical manufacture of monoclonal antibodies (mAbs),
purification is typically achieved using a series of chromatography
resins that are product-specific. Consequently the resins are often
used for just a few cycles, particularly if the therapeutic candidate
is unsuccessful where the resin is discarded before it has reached
its full potential cycle lifetime. As a result, chromatography
resin costs tend to dominate the material costs during clinical
manufacture. Continuous chromatographic systems offer the
ability to increase resin utilization and decrease the overall volume
of resin and associated manufacturing costs. This presentation will
explore the potential of generating early phase clinical material
via a continuous chromatographic system, before switching to
the conventional chromatography platform for late phase and
commercial production without product comparability concerns.
The presentation will also address the economic impact of operating
a continuous chromatographic system to generate clinical material
for a number of manufacturing scale and scheduling scenarios.
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BIOT 533 – 2:20 p.m.
One resin, multiple products: A green approach to purification
Ekta Mahajan, [email protected], Kapil Kothary.Department of Process
process economics, scale-up and tech transfer issues.
BIOT 535 – 3:00 p.m.
Development Engineering, Genentech, South San Francisco, CA 94080,
United States
Application of online bioreactor monitoring and feedback tools
for rapid development of better optimized cell culture processes
Protein A Affinity Chromatography is used for the purification of
monoclonal antibodies from harvested cell culture fluid (HCCF).
Typically, one column is dedicated for each MAb, which results
in resin being used to only 10% of its lifespan in pilot plant and
clinical production. However, significant savings can be realized
each year if resin is used for multiple products. In this study, a
cleaning procedure “MabSelect SuRe Campaign Changeover
Procedure (MSSCCP )” was developed at lab-scale that reduced
protein carryover to below assay detectable limits allowing use
of one column for multiple products. The re-use procedure was
successfully implemented on pilot plant columns in Oct 2010 used
for producing drug substance
Valerie Liu Tsang1, [email protected], An Zhang1,
BIOT 534 – 2:40 p.m.
Aqueous two-phase extraction in supramolecule purification
Yi Li, [email protected], Karan Mehra, Sianny Christanti, Mike Laksa.
Vaccine Process Development, Merck, West Point, PA 19486, United States
Although Aqueous Two-phase Extraction (ATPE) has been
studied with virtually all types of biomolecules, its application
in the pharmaceutical industry remains rare. This is partially
due to the large operational space where multiple mechanisms
can influence the partition simultaneously. Early stage process
development often solely depends on empirical screening, which
is less favored in comparison to other established methods.
However, supramolecules such as live virus, virus-like particles,
and membrane vesicles exhibit a similar partition behavior in
PEG-dextran system, where they almost exclusively partition
to the bottom dextran phase. This provides a potential platform
process development opportunity for recovery, concentration,
and purification in a single step. In addition, TECAN-based high
throughput screening, utilized to establish phase diagram and
impurity partition profiles, can further reduce the development
resources and timeline. In this work, experimental data with
multiple vaccine targets confirmed previous documented findings,
and demonstrated further benefits for aseptic operation and
biocontainment. More importantly, improved in-process stability
was observed in ATPE when compared to tangential flow filtration
(TFF). That directly translates to higher potency and operation
flexibility. Further comparison between ATPE and TFF in several
case studies is discussed regarding purification performance,
Marty Sinacore2, Thomas Ryll2. (1) Cell Culture Development, Biogen
Idec, Inc., Research Triangle Park, NC 27709, United States (2) Cell
Culture Development, Biogen Idec, Inc., Cambridge, MA 02142, United
States
Bioreactor feed strategies are typically developed over a series of
experiments using empirical offline data. Automatic bioreactor
sampling and feedback has been applied as a development tool
in which cell physiology dynamically determines the nutrient
requirements of the culture. The knowledge gained from this
work has then been translated to a less invasive monitoring and
feedback system in which platform feed strategies can be quickly
defined through the use of biocapacitance sensors and customized
bioreactor control code. This work will be discussed in the context
of shortening development timelines and improved process
optimization.
BIOT 536 – 3:40 p.m.
Biofabrication of on-chip bioprocessing stations toward
operational continuity
Jessica L Terrell1,2, [email protected], Tanya Gordonov1,2, Yi
Cheng3, Hsuan-Chen Wu1,2, Darryl Sampey1, Xiaolong Luo2, Chen-Yu
Tsao2, Reza Ghodssi4, Gary W Rubloff3,5, Gregory F Payne1,2, William
E Bentley1,2. (1) Fischell Department of Bioengineering, University
of Maryland, College Park, MD 20742, United States (2) Institute for
Bioscience and Biotechnology Research, University of Maryland, College
Park, MD 20742, United States (3) Institute for Systems Research,
University of Maryland, College Park, MD 20742, United States (4)
Department of Electrical and Computer Engineering, University of
Maryland, College Park, MD 20742, United States (5) Department of
Materials Science and Engineering, University of Maryland, College Park,
electrodeposited film pattern is programmable by the electrode’s
geometry. We have demonstrated the sequential deposition of
adjacent films and functionalization using a custom-engineered
protein in order to build stations for cell cultivation and
productivity monitoring. While the biofabricated stations are
physically separated onto non-contiguous electrodes within a single
“chip”, interaction occurs between components at each station by
diffusion across pliable boundaries. Innovative means that enable
complete binding reversibility allow for subsequent cell and protein
recovery. We believe biofabrication strategies such as this “in-film”
bioprocessing approach are simple and rapid tools that will enable
miniaturization of clonal analysis for rapid selection at a reduced
yet augmentable scale for optimal production.
BIOT 537 – 4:00 p.m.
Computational fluid dynamics for bio-reactors: Opportunities
to accelerate process development
Damodaran Vedapuri2, [email protected], Gopal R
Kasat1. (1) Tridiagonal Solutions Pvt. Ltd, Pune, Maharashtra 411008,
India (2) Tridiagonal Solutions Inc, San Antonio, Texas 78230, United
States
Design of bioreactors for large scale production is complex and
time consuming. Experimental testing in lab and pilot, are both
time consuming and expensive and does not always help map the
underlying influence of reactor hardware and operating protocols
on process yields. Alternatively, computational fluid dynamics
(CFD) technology can be efficiently employed to develop a deeper
understanding of the complex interaction between the reactor
hardware, the process environment and the process yields. This
study aims at discussing various case studies where CFD technology
was successfully employed for process improvement and scale-up.
The first case study will focus on use of CFD methodology for
the efficient scale-up of a bioreactor. The second case study will
highlight use of CFD for design modification. The third case study
will focus on use of CFD methods to predict reactor performance.
The fourth case study will highlight use CFD methodologies for
modifying existing hardware.
MD 20742, United States
Some of the major advances in bioprocessing can be attributed
to the miniaturization of hardware components for smallscale operation; yet, integrating multiple operations remains a
challenge because of the difficulty in allocating the labile biological
components within spatial boundaries. To address this challenge,
we have used “biofabrication” to organize biomolecules and cells
at a millimeter scale. Specifically, we have employed stimuliresponsive polysaccharides, film-forming in the presence of an
electrical signal, to assemble films onto electrode surfaces. The
(BIOT 538 on following page)
191
Thursday Afternoon
the growth, viability, and productivity of the cells. In addition,
endogenous UPR activation was significantly impacted by the cell
culture environment, i.e. amino acid levels and osmolarity. We
will discuss the role of UPR-mediated transcriptional activation
of different signaling pathways on cell performance during
recombinant protein production, and the use of an inducible
system and UPR monitoring to engineer or improve control of
recombinant protein production.
BIOT 538 – 4:20 p.m.
Systems biotechnology for drug discovery and development
Vikramaditya G Yadav, [email protected], Ajikumar P Kumaran,
Chin G Lim, Gregory N Stephanopoulos.Department of Chemical
Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139,
The ACS Biochemical Technology Division
gratefully acknowledges the financial support provided
by these biotechnology industry leaders.
United States
A major reason for the declining prospects of Big Pharma
is that synthetic chemistry cannot efficiently access more
‘druggable’ chemical space (e.g. natural products). Conversely,
other alternatives that can access natural product space, such as
metabolic engineering, are also restricted. An overwhelming
majority of the natural product pathways are uncharacterized, this
despite the staggering volume of biological information that is now
available. Methodologies that piece together information on genes,
enzymes and metabolites from a native producer into a coherent
metabolic pathway that can then be engineered into microbial
hosts do not exist. We have formulated an algorithm that achieves
this exact aim. The algorithm promises to considerably expand the
utility of metabolic engineering for synthesizing natural products
and their analogues. Moreover, we also propose a high-throughput
drug discovery platform that provides unprecedented access to
highly-druggable chemical space; conducts faster SAR studies and
lead optimization; and, significantly, necessitates no infrastructure
substitution.
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