Cell Line Development and Engineering
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June 21 - 23, 2010 · San Francisco Airport Marriott · Burlingame, CA
Document Title
Agenda
Main Conference: Monday | Main Conference: Tuesday | Main Conference: Wednesday
8:00
Chairperson's Opening Remarks
Kevin M. McCarthy, Ph.D., Principal Research Scientist, Drug Substance Development, Pfizer Biotherapeutics
Kevin M. McCarthy, Ph.D., Principal Research Scientist, Drug Substance Development, Pfizer Biotherapeutics
Advances in Understanding Cell Line Stability
Mammalian cell lines actively shut down expression of exogenously expressed genes as a defense mechanism. Topics in the session "Advances in Understanding Cell Line Stability" will speak to actions taken in our industry to manage or manipulate this defense mechanism. Some avenues pursued include the construction of expression vectors to alleviate instability, targeted integration into host cells and control of epigenetic factors to shut down gene expression.
8:15
Genetic (In)Stability in Recombinant Mammalian Cell Lines - The Nucleus as a Homeostatic Regulator
Tremendous advances have been made in vector systems (and associated delivery and selection) for high-level transgene expression in recombinant mammalian cell lines. Current challenges revolve around the complexity of the interaction of nuclear structures with assured stable and high-level transgene expression. Case studies (with CHO and myeloma cells) will be discussed in relation to current understanding and the future opportunities.
Alan Dickson, Ph.D., Professor of Biotechnology, Director, Center of Excellence for Biopharmaceuticals, The University of Manchester, United Kingdom
Tremendous advances have been made in vector systems (and associated delivery and selection) for high-level transgene expression in recombinant mammalian cell lines. Current challenges revolve around the complexity of the interaction of nuclear structures with assured stable and high-level transgene expression. Case studies (with CHO and myeloma cells) will be discussed in relation to current understanding and the future opportunities.
Alan Dickson, Ph.D., Professor of Biotechnology, Director, Center of Excellence for Biopharmaceuticals, The University of Manchester, United Kingdom
8:45
Mechanisms of Production Instability in CHO Cells
Clonal genetic variation is necessarily exploited to generate productive cell lines. However, spontaneously arising (epi)genetic heterogeneity also contributes to unpredictable and uncontrollable loss of cell specific production rate. Using CHO cell lines that exhibit either short or long term loss of productivity as examples I will describe the molecular basis of instability and discuss how this problem may be controlled.
David C. James, Ph.D., Professor of Bioprocess Engineering, Department of Chemical and Process Engineering, University of Sheffield, United Kingdom
Clonal genetic variation is necessarily exploited to generate productive cell lines. However, spontaneously arising (epi)genetic heterogeneity also contributes to unpredictable and uncontrollable loss of cell specific production rate. Using CHO cell lines that exhibit either short or long term loss of productivity as examples I will describe the molecular basis of instability and discuss how this problem may be controlled.
David C. James, Ph.D., Professor of Bioprocess Engineering, Department of Chemical and Process Engineering, University of Sheffield, United Kingdom
9:15
EESYR: An Integrated Approach for Increasing Capacity and Speed Through Early Phase Development of Recombinant Antibodies
Protein therapeutic production platforms are designed to increase throughput and speed to clinical development by minimizing process development. The essential component of any production platform is predictable cell line performance. Desirable platform cell line properties will be discussed and the EESYR targeted integration expression system will be contrasted with random integration methods.
James Fandl, Ph.D., Vice President, Protein Expression Sciences, Regeneron Pharmaceuticals, Inc.
Protein therapeutic production platforms are designed to increase throughput and speed to clinical development by minimizing process development. The essential component of any production platform is predictable cell line performance. Desirable platform cell line properties will be discussed and the EESYR targeted integration expression system will be contrasted with random integration methods.
James Fandl, Ph.D., Vice President, Protein Expression Sciences, Regeneron Pharmaceuticals, Inc.
9:45
Vector Redesign to Improve Cell Line Stability and Clone Selection
Abstract not available at press date.
Kevin M. McCarthy, Ph.D., Principal Research Scientist, Drug Substance Development, Pfizer Biotherapeutics
Abstract not available at press date.
Kevin M. McCarthy, Ph.D., Principal Research Scientist, Drug Substance Development, Pfizer Biotherapeutics
10:15
Networking Refreshment Break in Exhibit/Poster Hall
Genomics and Beyond - New Techniques & Technologies to Optimize Cell Line Development
'Omics entered the biotechnology field with lots of fanfare and promise, and several uses are bearing fruit. In the session "Genomics and Beyond- New Techniques and Technologies to Optimize Cell Line Development," we will see examples of how investigating cell metabolism leads to a better understanding of cell physiology and how similar metabolic analysis can be used to optimize cell culture conditions. We shall also hear about how advances in genomics are leading to practical applications, such as gene knockouts, to create better production lineages.
10:45
Robust Cell Culture Process Development Using Metabolic Flux Analysis
Cell line and process development decisions in commercial mammalian cell culture are typically based on prime variable measurements and subsequent computation of cell specific rates. While useful, this data set does not provide a comprehensive description of the cell physiological state and metabolic flux analysis is one approach which provides additional information on cell physiology and metabolism. Application of metabolic flux analysis to cell line and process development will be presented.
Chetan Goudar, Ph.D., Head, Cell Culture Development, Global Biological Development, Bayer HealthCare
Cell line and process development decisions in commercial mammalian cell culture are typically based on prime variable measurements and subsequent computation of cell specific rates. While useful, this data set does not provide a comprehensive description of the cell physiological state and metabolic flux analysis is one approach which provides additional information on cell physiology and metabolism. Application of metabolic flux analysis to cell line and process development will be presented.
Chetan Goudar, Ph.D., Head, Cell Culture Development, Global Biological Development, Bayer HealthCare
11:15
Case
Study
Use of Metabolomics Time Course Analysis for Media OptimizationStudy
Maximizing the production of biologicals in cell culture systems is a highly empirical process that requires multiple rounds of cause and effect analysis centering on productivity and quality as the end point drivers. Historically a limited number of metabolites are relied on to provide insight into the metabolic state of the cell and the performance of the culture conditions. A powerful new tool for extending this intuitive link between phenotypic output and metabolic changes is global unbiased metabolic profiling. Comprehensive biochemical profiling analyzes the change in hundreds of biochemicals in the cells and conditioned media.
Michael Milburn, Ph.D., Chief Scientific Officer, Metabolon
11:45
CHO Bioproduction GeneTarget Discovery and Cell Line Creation Using Zinc Finger Nuclease Technology
In an effort to define the molecular characteristics of high-producing CHO cells, we designed an experiment that would enable us to identify genes that are differentially expressed upon a significant decline in the metabolic burden of recombinant IgG protein production. We designed small interfering RNAs (siRNAs) against heavy and light chains of a recombinant human IgG, and transfected these siRNAs into a highly productive CHO cell line stabling expressing this IgG. Treatment with IgG siRNAs led to differential expression of numerous genes involved in protein secretion and folding, immunoglobulin production and protein glycosylation. The results reported here may shed light on target selection in systems biology directed cell line engineering of CHO cells. I will chronicle several case studies where ZFN technology was used to create KO mutants in CHO cell lines.
Kevin Kayser, Ph.D., R&D Director, Cell Sciences and Development, SAFC
In an effort to define the molecular characteristics of high-producing CHO cells, we designed an experiment that would enable us to identify genes that are differentially expressed upon a significant decline in the metabolic burden of recombinant IgG protein production. We designed small interfering RNAs (siRNAs) against heavy and light chains of a recombinant human IgG, and transfected these siRNAs into a highly productive CHO cell line stabling expressing this IgG. Treatment with IgG siRNAs led to differential expression of numerous genes involved in protein secretion and folding, immunoglobulin production and protein glycosylation. The results reported here may shed light on target selection in systems biology directed cell line engineering of CHO cells. I will chronicle several case studies where ZFN technology was used to create KO mutants in CHO cell lines.
Kevin Kayser, Ph.D., R&D Director, Cell Sciences and Development, SAFC
Technology Workshop
12:15
This workshop will demonstrate new capabilities for mammalian cell culture process development using the Micro-24 Microbioreator system. We will illustrate the use of a new disposable bioreactor cassette for mammalian applications and describe how to rapidly implement this new technology in a cell culture process development facility. We will show how the Micro-24 technology simplifies reactor preparation workflow, dramatically reduces set-up time (from days to minutes), enables rapid, easy feeding and sampling, and automates reactor control and data acquisition.
Tiffany Rau, Ph.D., Pall Corporation
12:45
Networking Luncheon in Exhibit/Poster Hall
2:00
Chairperson's Remarks
John H. Chon, Ph.D., Senior Director, BioProcess R&D, Percivia, LLC
John H. Chon, Ph.D., Senior Director, BioProcess R&D, Percivia, LLC
Screening and Clone Selection
An accurate and predictive method for screening clones is a critical component in any cell line generation program. The session "Screening and Clone Selection" will consist of presentations that will discuss the various systems specifically designed to assess and select clones that will perform well in large scale manufacturing processes. We will hear about the development and application of multi-well plate-based clone screening techniques, as well as how FACS and automations have been used to select more productive clones while reducing project timelines.
2:15
Accelerated Timelines, Reduced Resource Requirement and High Quality Cell Lines Delivered by Implementation of FACS in a CHO Cell Culture Fed-Batch Process
By using a combination of FACS technology to isolate highly productive clonal cell lines and screening the clones using a platform process we have decreased the project timelines and enabled efficient use of resources for delivery of a CHO fed-batch process capable of delivering target titers.
Delfi Krishna, Ph.D., Principal Scientist, Microbial and Cell Culture Development, GlaxoSmithKline
By using a combination of FACS technology to isolate highly productive clonal cell lines and screening the clones using a platform process we have decreased the project timelines and enabled efficient use of resources for delivery of a CHO fed-batch process capable of delivering target titers.
Delfi Krishna, Ph.D., Principal Scientist, Microbial and Cell Culture Development, GlaxoSmithKline
2:45
Use of High Throughput Fed-Batch Culture: Impact on Cell Line Constructions
Screening cell lines for productivity using a fed-batch culture process is an integral part of constructing cell lines suitable for cGMP manufacture. This presentation will review some of the technologies that Lonza has evaluated for scale down fed-batch culture and present data on how a shaking 96-well plate fed-batch screen is incorporated into Lonza's One-Step cell line construction process.
Adrian Haines, Ph.D., Senior Group Leader, Cell Culture Process Development, Lonza Biologics, United Kingdom
Screening cell lines for productivity using a fed-batch culture process is an integral part of constructing cell lines suitable for cGMP manufacture. This presentation will review some of the technologies that Lonza has evaluated for scale down fed-batch culture and present data on how a shaking 96-well plate fed-batch screen is incorporated into Lonza's One-Step cell line construction process.
Adrian Haines, Ph.D., Senior Group Leader, Cell Culture Process Development, Lonza Biologics, United Kingdom
3:15
Networking Refreshment Break in Exhibit/Poster Hall
3:45
High-Throughput Fed Batch Suspension Productivity Screening in MAb Expression Clone Selection
Large numbers of clones must be screened to increase the probability of finding cell lines capable of producing high titers of monoclonal antibodies. Static titers have negligible predictive value for shake flask or bioreactor productivity. We will discuss results obtained using optimized conditions for shaken deepwell plates coupled with an automated liquid handling system to identify high producers more efficiently.
Pamela Hawley-Nelson, Ph.D., Associate Director, Process Cell Culture, MedImmune
Large numbers of clones must be screened to increase the probability of finding cell lines capable of producing high titers of monoclonal antibodies. Static titers have negligible predictive value for shake flask or bioreactor productivity. We will discuss results obtained using optimized conditions for shaken deepwell plates coupled with an automated liquid handling system to identify high producers more efficiently.
Pamela Hawley-Nelson, Ph.D., Associate Director, Process Cell Culture, MedImmune
4:15
Case
Study
Development of CHO Production Cell Lines Using the ClonePix FLStudy
Abstract not available at press date.
Stephanie E. Rieder, Ph.D., Senior Scientist, Abbott Bioresearch Center
4:45
Development of the Screening Methods Using a Micro24 Scale Down Bioreactor - Good High Throughput Model for Bioreactors
Screening cell lines and developing processes are traditionally initiated in relatively uncontrolled environments which poorly model bioreactors. A robust and scalable method of screening in a controlled environment was developed using the Micro-24 Bioreactor. "High-throughput" bioreactors increase the likelihood of selecting cell lines and developing processes that are "winners" from bench top to commercialization with fewer resources and shorter timelines.
Tiffany D. Rau, Ph.D., Principal Scientist, Microbial and Cell Culture Development, GlaxoSmithKline
Screening cell lines and developing processes are traditionally initiated in relatively uncontrolled environments which poorly model bioreactors. A robust and scalable method of screening in a controlled environment was developed using the Micro-24 Bioreactor. "High-throughput" bioreactors increase the likelihood of selecting cell lines and developing processes that are "winners" from bench top to commercialization with fewer resources and shorter timelines.
Tiffany D. Rau, Ph.D., Principal Scientist, Microbial and Cell Culture Development, GlaxoSmithKline
5:15
Cocktail Reception in Exhibit/Poster Hall
Sponsored by
Sponsored by
Main Conference: Monday | Main Conference: Tuesday | Main Conference: Wednesday
8:00
Chairperson's Opening Remarks
John Joly, Ph.D., Director of Early Stage Cell Culture, Process Development, Genentech, Inc.
John Joly, Ph.D., Director of Early Stage Cell Culture, Process Development, Genentech, Inc.
Plenary Session - What Comes Next After Titer Increase?
This session will tackle the following: 1) futuristic views for cell line engineering and cell culture process development, 2) production of difficult recombinant proteins and any insights trying to avoid such problematic molecular characteristics, and 3) if you can produce high titers and create challenges for the purification group, what strategies are employed by your downstream partners to cope with the increased batch sizes.
Keynote Address
8:05
Are There Drivers Left for Further Process Optimization in Cell Line Development?Now that production titers in excess of > 5 g/L are being routinely reported, this address will examine other manufacturing attributes that could drive additional cell culture process development. Such drivers that will be examined include: (i) manufacturing cost; (ii) development cycle times; (iii) raw material quality assurance; (iv) standardization; (v) growth rate; (vi) design space; (vii) impurities; (viii) regulatory relief.
Michael W. Glacken, Ph.D., Senior Director, Biologics Process Development, Millennium Pharmaceuticals
Keynote Address
8:40
Beyond Just High Titers - The Future of Cell Line EngineeringThe breadth of cell line engineering is expanding to more directly address the following goals:
- Reduced variability in titers between different products
- Reduced levels of contaminating host cell proteins
- Simpler downstream processing
- Superior glycoforms
- Faster and cheaper overall process and product development
Matthew S. Croughan, Ph.D., George B. and Joy Rathmann Professor and Director of the Amgen Bioprocessing Center, Keck Institute
Featured Presentation
9:15
Difficult-to-Express Proteins and Using Transient Expression to Predict Stable Cell Line Behavior
Abstract not available at press date.
John Delaney, Ph.D., Director, Protein Sciences, Amgen Inc.
Abstract not available at press date.
John Delaney, Ph.D., Director, Protein Sciences, Amgen Inc.
Featured Presentation
9:45
Strategies for Resolving Downstream Bottlenecks to Enable High Titer Recoveries
Increases in cell culture titer have the potential to create downstream process bottlenecks. These bottlenecks can be alleviated to some extent by employing current downstream process platforms with high capacity resins and carefully arranged process steps to minimize pool volumes. Beyond some facility specific, maximum titer, introduction of new process technology must be considered if full recovery of high titer bulks is required.
Chris Dowd, Ph.D., Principal Engineer, Senior Group Leader, Late Stage Purification, Genentech, Inc.
Increases in cell culture titer have the potential to create downstream process bottlenecks. These bottlenecks can be alleviated to some extent by employing current downstream process platforms with high capacity resins and carefully arranged process steps to minimize pool volumes. Beyond some facility specific, maximum titer, introduction of new process technology must be considered if full recovery of high titer bulks is required.
Chris Dowd, Ph.D., Principal Engineer, Senior Group Leader, Late Stage Purification, Genentech, Inc.
10:15
Networking Refreshment Break in Exhibit/Poster Hall
Utility of Statistical Tools in Cell Line & Process Development
This session will focus on how to get the most out of large data sets that both cell line development and media development can generate. With medium and high throughput systems large amounts of data can overwhelm users and obfuscate meaningful trends. These presentations will illuminate how proper design and data treatment can shed important insights with rapid turnaround.
10:45
Application of Deep Well Plates and DOE to Clone Screening Experiments
Traditional, low throughput screening approaches have hampered our ability to thoroughly screen and characterize a large number of clones. We have developed a deep well plate based protocol that has increased our throughput for identifying top clones. Using this new format we are able to apply DOE to clone screening experiments and help identify important factors for clone performance.
Allison Bianchi, Ph.D., Senior Scientist, Cell Science and Technology, Amgen Inc.
Traditional, low throughput screening approaches have hampered our ability to thoroughly screen and characterize a large number of clones. We have developed a deep well plate based protocol that has increased our throughput for identifying top clones. Using this new format we are able to apply DOE to clone screening experiments and help identify important factors for clone performance.
Allison Bianchi, Ph.D., Senior Scientist, Cell Science and Technology, Amgen Inc.
11:15
DOE Approach - Media Design and Development Using Statistical Tools - What have We Learned?
Historically, mammalian cell culture media design and development has primarily involved the empirical determination of optimal media compositions. Although the large number of interacting components makes media optimization experimentally complex and time consuming, statistical approaches can significantly reduce this complexity and allow one to quickly achieve optimal media formulations.
Brian D. Follstad, Ph.D., Senior Scientist, Cell Science and Technology, Amgen Inc.
Historically, mammalian cell culture media design and development has primarily involved the empirical determination of optimal media compositions. Although the large number of interacting components makes media optimization experimentally complex and time consuming, statistical approaches can significantly reduce this complexity and allow one to quickly achieve optimal media formulations.
Brian D. Follstad, Ph.D., Senior Scientist, Cell Science and Technology, Amgen Inc.
Audience Interactive Panel Discussion
11:45
Sponsored by
Challenges & Advantages of Using the GS Gene Expression SystemModerator: Andy Racher, Ph.D., Senior Principal Scientist, Lonza Biologics, United Kingdom
Panelists:
Lianchun Fan, Ph.D., Research Scientist, Bioprocess R&D, Eli Lilly and Company
Paul Sauer, Senior Director, Development, Oncomed Pharmaceuticals
Kurt Droms, Ph.D., Associate Research Fellow, BioTherapeutics Pharmaceutical Sciences, BioTherapeutics Research and Development, Pfizer Corporation
12:30
Networking Luncheon in Exhibit/Poster Hall
1:30
Chairperson's Remarks
Pamela Hawley-Nelson, Ph.D., Associate Director, Process Cell Culture, MedImmune
Pamela Hawley-Nelson, Ph.D., Associate Director, Process Cell Culture, MedImmune
Addressing Timeline Bottlenecks in Development
Timelines for mammalian cell line development and the downstream processes, formulations and analytical tests that are required before manufacturing and IND application are not always synchronized. Topics in the session "Addressing Timeline Bottlenecks in Development" will speak to the balancing required in projects to optimize resources and timing. Talks will cover cell line development, antibody production and the utility of platform approaches.
1:45
Developing a Rapid Cell Line Construction Process to Deliver Research Materials Quickly
Abstract not available at press date.
Timothy Gryseels, M.A., Senior Scientist, Global Biologics, Pfizer
Abstract not available at press date.
Timothy Gryseels, M.A., Senior Scientist, Global Biologics, Pfizer
2:15
VelociMabTM Technology for Isolation and Production of Therapeutic Antibodies
VelociMabTM is a suite of Regeneron's proprietary technologies that integrate high throughput antibody isolation from B cells, construction of IND-enabling expression cell lines, and optimized bioreactor production process. Best-in-class VelociMabTM antibodies have been isolated against diverse targets. VelociMabTM cell lines readily produce antibodies at a level greater than one gram per liter in bioreactors. VelociMabTM technology significantly reduces development time of therapeutic antibodies.
Gang Chen, Ph.D., Senior Director, Cell Technologies, Regeneron Pharmaceuticals, Inc.
VelociMabTM is a suite of Regeneron's proprietary technologies that integrate high throughput antibody isolation from B cells, construction of IND-enabling expression cell lines, and optimized bioreactor production process. Best-in-class VelociMabTM antibodies have been isolated against diverse targets. VelociMabTM cell lines readily produce antibodies at a level greater than one gram per liter in bioreactors. VelociMabTM technology significantly reduces development time of therapeutic antibodies.
Gang Chen, Ph.D., Senior Director, Cell Technologies, Regeneron Pharmaceuticals, Inc.
2:45
Process Platformization to Accelerate Timeline to Clinical Manufacturing
Platform approach in defining cell culture process for clinical manufacturing has gained a lot of momentum in biopharmaceutical development of early stage biologics. Established process platform promotes cost saving on resources and raw materials as well as significant reduction in development timeline while ensuring acceptable yield and product quality. This presentation will discuss how process parameters across cell culture unit operations, from vial thaw to harvest, can be platformized and highlights when or where flexibility may be required.
Helena Y. Makagiansar, Ph.D., Associate Director, Biopharmaceutical Development, Biogen Idec
Platform approach in defining cell culture process for clinical manufacturing has gained a lot of momentum in biopharmaceutical development of early stage biologics. Established process platform promotes cost saving on resources and raw materials as well as significant reduction in development timeline while ensuring acceptable yield and product quality. This presentation will discuss how process parameters across cell culture unit operations, from vial thaw to harvest, can be platformized and highlights when or where flexibility may be required.
Helena Y. Makagiansar, Ph.D., Associate Director, Biopharmaceutical Development, Biogen Idec
3:15
Networking Refreshment Break in Exhibit/Poster Hall
Improving Fundamental Understanding /Strategies for Modifying or New Parental Lines
Host cell lines provide the critical machinery for expressing therapeutic proteins. Studies of cell biology have suggested avenues to increase the effectiveness and longevity of that machinery. Topics in the session "Improving Fundamental Understanding / Strategies for Modifying or New Parental Cell Lines" will speak to utility of viral proteins to activate useful pathways, the contribution of over-expressed anti-apoptotic genes to longevity, butyrate tolerance and productivity, engineering host cell phenotypes such as lactate dehydrogenase activity by siRNA treatment and identifying other potential targets using miRNA profiles.
3:45
CHO Host Cell Engineering Using Viral Genes
Selected viral proteins may activate a number of cellular pathways that could be advantageous for a host used in the production of heterologous proteins. A comparison of antibody producing clones from transfections using the original CHO host versus clones derived from a CHO host engineered to express polyoma T-antigens (TAg host) will be presented. Preliminary data suggests use of the TAg host can lead to clones with increased productivity.
Laura Simmons, Ph.D., Scientist, Department of Early Stage Cell Culture, Genentech, Inc.
Selected viral proteins may activate a number of cellular pathways that could be advantageous for a host used in the production of heterologous proteins. A comparison of antibody producing clones from transfections using the original CHO host versus clones derived from a CHO host engineered to express polyoma T-antigens (TAg host) will be presented. Preliminary data suggests use of the TAg host can lead to clones with increased productivity.
Laura Simmons, Ph.D., Scientist, Department of Early Stage Cell Culture, Genentech, Inc.
4:15
Expression of Anti-Apoptotic Proteins in Commercially-Relevant Mammalian Cells to Increase Viability and Production of Recombinant Biotherapeutics
In an effort to develop robust Chinese Hamster Ovary host cell lines, a variety of anti-apoptotic genes were over-expressed, either singly or in combination, followed by high-throughput screening of transfectants for improved cell growth, extended longevity, reduced caspase 3/7 activity and tolerance to sodium butyrate. The resulting apoptotic-resistant cell lines exhibited significant metabolic differences compared to normal cell lines, a knowledge that can be utilized in developing superior production cell lines expressing biotherapeutics.
Haimanti Dorai, Ph.D., Senior Research Fellow in Pharmaceutical Development, Centocor R&D
In an effort to develop robust Chinese Hamster Ovary host cell lines, a variety of anti-apoptotic genes were over-expressed, either singly or in combination, followed by high-throughput screening of transfectants for improved cell growth, extended longevity, reduced caspase 3/7 activity and tolerance to sodium butyrate. The resulting apoptotic-resistant cell lines exhibited significant metabolic differences compared to normal cell lines, a knowledge that can be utilized in developing superior production cell lines expressing biotherapeutics.
Haimanti Dorai, Ph.D., Senior Research Fellow in Pharmaceutical Development, Centocor R&D
4:45
The Taming of CHO Cell Protein Expression Using RNAi
Silencing RNAs (siRNAs) can decrease the cellular production of targeted proteins by promoting specific degradation of their messenger RNA. In an analogous manner, siRNAs can be used in bioprocessing to down regulate expression of undesired genes. By media additions of potent siRNAs with pico-molar IC50 values directed against hamster apoptotic and metabolism genes (Bax, Bak, and lactate dehydrogenase), we were able to affect the growth of Chinese Hamster Ovary (CHO) cells grown in suspension under bioprocessing batch-fed conditions when compared to control siRNA-treated cells. These siRNAs were dosed at 1 nM concentrations and CHO cells were monitored for cell growth, apoptosis activity, and the production of lactate. These doses of siRNA significantly reduced mRNA levels of their respective genes by greater than 80-90% with a corresponding decrease in lactate dehydrogenase activity and a decrease in mitochondrial apoptotic pathway activity as measured by decreased caspase 3 activity. Integral cell areas nearly doubled in the Bax/Bak/LDH siRNA-treated cultures. Additional CHO cellular pathways are currently being investigated using siRNAs directed against critical glycosylation enzymes and protein production/translation pathways.
Anthony (Tony) Rossomando, Ph.D., Senior Director, Alnylam Biotherapeutics
Silencing RNAs (siRNAs) can decrease the cellular production of targeted proteins by promoting specific degradation of their messenger RNA. In an analogous manner, siRNAs can be used in bioprocessing to down regulate expression of undesired genes. By media additions of potent siRNAs with pico-molar IC50 values directed against hamster apoptotic and metabolism genes (Bax, Bak, and lactate dehydrogenase), we were able to affect the growth of Chinese Hamster Ovary (CHO) cells grown in suspension under bioprocessing batch-fed conditions when compared to control siRNA-treated cells. These siRNAs were dosed at 1 nM concentrations and CHO cells were monitored for cell growth, apoptosis activity, and the production of lactate. These doses of siRNA significantly reduced mRNA levels of their respective genes by greater than 80-90% with a corresponding decrease in lactate dehydrogenase activity and a decrease in mitochondrial apoptotic pathway activity as measured by decreased caspase 3 activity. Integral cell areas nearly doubled in the Bax/Bak/LDH siRNA-treated cultures. Additional CHO cellular pathways are currently being investigated using siRNAs directed against critical glycosylation enzymes and protein production/translation pathways.
Anthony (Tony) Rossomando, Ph.D., Senior Director, Alnylam Biotherapeutics
5:15
Engineering CHO Production Phenotypes by Specific Modification of MicroRNA Expression Levels
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. They have been implicated in diverse cellular functions, including proliferation and protein secretion, and are currently the subject of considerable interest in all aspects of cell biology. Using expression profiling we have identified several miRNAs as potential engineering targets to modify CHO cell behavior in culture.
Niall Barron, Ph.D., Senior Research Scientist, National Institute for Cellular Biotechnology, Dublin City University, Ireland
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. They have been implicated in diverse cellular functions, including proliferation and protein secretion, and are currently the subject of considerable interest in all aspects of cell biology. Using expression profiling we have identified several miRNAs as potential engineering targets to modify CHO cell behavior in culture.
Niall Barron, Ph.D., Senior Research Scientist, National Institute for Cellular Biotechnology, Dublin City University, Ireland
5:45
Close of Day Two
Main Conference: Monday | Main Conference: Tuesday | Main Conference: Wednesday
8:00
Chairperson's Opening Remarks
Kevin Kayser, Ph.D., R&D Director, Cell Sciences and Development, SAFC
Kevin Kayser, Ph.D., R&D Director, Cell Sciences and Development, SAFC
Approaches to Improve Product Quality
Factors such as glycosylation variability, amino acid mis-incorporation and antibody disulfide bond reduction are all important parameters to examine when assessing biopharmaceutical product quality. Topics in this session "Approaches to Improve Product Quality" examine product quality challenges and solutions in our industry today.
8:15
Case
Study
Unexpected Product Quality ChallengesStudy
During production of a monoclonal antibody, reduction of the antibody's interchain disulfide bonds was observed. This antibody reduction event was catastrophic as the product failed to meet the drug substance specifications and the bulk product was lost. Several methods were tested to prevent antibody reduction and will be presented here. Additional examples of product quality challenges may also be discussed.
Michael Laird, Ph.D., Associate Director - Senior Scientist, Late Stage Cell Culture, Genentech, Inc.
8:45
Case
Study
Discovery and Investigation of Mis-Incorporation of Serine at Asparagine Positions in Recombinant Proteins Expressed in CHO CellsStudy
Multiple CHO cell lines were developed expressing an antibody. During antibody analysis from early stage fed-batch cultures, detectable levels of serine residues were found in random positions coding for asparagine. A statistical evaluation of the cell lines and culture conditions identified that the presence of misincorporated serine residues in the secreted antibody correlated most strongly with the concentration of asparagine in the culture medium. Experimental studies were conducted to intentionally and reproducibly introduce or minimize the levels of misincorporation.
Jana Dolnikova, M.S., Senior Associate Scientist, Clinical Cellular Engineering, Biogen Idec
9:15
Identification of Cell Culture Conditions to Control Glycosylation of Recombinant Glycoproteins Expressed in CHO Cells
Because each cell culture process may be unique, development of glycosylation control strategies is often needed to ensure product comparability through the development cycle and ultimately enable manufacturing consistency. Glycosylation variability may be attributed to cell line or culture conditions (e.g., temperature, pH, media). To better understand these effects on glycosylation, correlations of process conditions with glycoforms are summarized for several cell lines.
Kara Calhoun, Ph.D., Senior Engineer, Late Stage Cell Culture, Genentech, Inc.
Because each cell culture process may be unique, development of glycosylation control strategies is often needed to ensure product comparability through the development cycle and ultimately enable manufacturing consistency. Glycosylation variability may be attributed to cell line or culture conditions (e.g., temperature, pH, media). To better understand these effects on glycosylation, correlations of process conditions with glycoforms are summarized for several cell lines.
Kara Calhoun, Ph.D., Senior Engineer, Late Stage Cell Culture, Genentech, Inc.
9:45
Carbohydrate Engineering to Improve the Efficacy of Recombinant Proteins
Glycosylation plays an important role in the efficacy of many recombinant proteins, affecting stability, half-life cellular uptake, tissue targeting and potentially immunogenicity. When glycosylation is less than optimal there are a variety of strategies which can be utilized to improve these biological properties, including adding or removing glycosylation sites, adding or removing individual sugar residues, engineering or selecting mutant cell lines to modify expression of glycosyltransferases, metabolically modifying glycosylation and conjugating neoglycans to proteins. Examples of each approach will be discussed.
Tim Edmunds, Ph.D., Vice President, Therapeutic Protein Research, Genzyme Corporation
Glycosylation plays an important role in the efficacy of many recombinant proteins, affecting stability, half-life cellular uptake, tissue targeting and potentially immunogenicity. When glycosylation is less than optimal there are a variety of strategies which can be utilized to improve these biological properties, including adding or removing glycosylation sites, adding or removing individual sugar residues, engineering or selecting mutant cell lines to modify expression of glycosyltransferases, metabolically modifying glycosylation and conjugating neoglycans to proteins. Examples of each approach will be discussed.
Tim Edmunds, Ph.D., Vice President, Therapeutic Protein Research, Genzyme Corporation
10:15
Networking Refreshment Break
Large Scale Transient Transfection
Process development scientists have been using transient transfection and stable pools to rapidly generate r-proteins and antibodies to use in research, IND, and tox studies. The session "Large Transient Transient Transfection" will review methods to develop, optimize and implement processes to generate intermediate quantities of therapeutic and research proteins.
10:45
Case
Study
High Yielding CHO Transient System for Producing Preclinical Grade Recombinant ProteinStudy
An efficient, rapid and productive expression strategy is required to meet the increasing requirements for research grade material during early drug development. The development, optimisation and implementation of a scalable high yielding proprietary CHO transient expression system would be presented. The system has been successfully scaled up to 250L and is capable of expressing several hundred mg/L of recombinant protein. This CHO transient system can now provide gram amounts of preclinical grade material.
Lekan Daramola, Ph.D., Associate Director, Early Expression and Supply, Cell Sciences, Development, MedImmune Ltd, United Kingdom
11:15
Optimization of CHO Transient Transfection Systems for Protein Production
Transient transfection of CHO cells is commonly used for rapid production of recombinant proteins. This presentation will discuss various improvements to our transient transfection system including host cell engineering and process development. The result is a flexible, high throughput protein production system capable of accommodating a broad range of product demand within the tight timelines required for research and therapeutic studies.
Athena Wong, Ph.D., Associate Scientist, Early Stage Cell Culture, Genentech, Inc.
Transient transfection of CHO cells is commonly used for rapid production of recombinant proteins. This presentation will discuss various improvements to our transient transfection system including host cell engineering and process development. The result is a flexible, high throughput protein production system capable of accommodating a broad range of product demand within the tight timelines required for research and therapeutic studies.
Athena Wong, Ph.D., Associate Scientist, Early Stage Cell Culture, Genentech, Inc.
11:45
Alternatives to Large-Scale Transient Transfection for Rapid Expression of Antibodies and Bispecific DART Molecules
While transient transfections can provide recombinant proteins in a relatively short amount of time, there are also drawbacks such as large culture volumes and the need for greater amounts of DNA and reagents. As an alternative and more economical means to produce intermediate amounts of recombinant protein we have developed effective stable pool strategies. These methods requires microgram amounts of DNA and can rapidly generate 50-400mg/L. Additional batches of protein can be made without repeated DNA preparation and transfection. We have optimized these methods in suspension-adapted CHO cells with the GS and UCOE vector systems for the expression of antibodies and bispecific DART molecules.
Valentina C. Ciccarone, Ph.D., Principal Scientist, Cell Line Development, MacroGenics, Inc.
While transient transfections can provide recombinant proteins in a relatively short amount of time, there are also drawbacks such as large culture volumes and the need for greater amounts of DNA and reagents. As an alternative and more economical means to produce intermediate amounts of recombinant protein we have developed effective stable pool strategies. These methods requires microgram amounts of DNA and can rapidly generate 50-400mg/L. Additional batches of protein can be made without repeated DNA preparation and transfection. We have optimized these methods in suspension-adapted CHO cells with the GS and UCOE vector systems for the expression of antibodies and bispecific DART molecules.
Valentina C. Ciccarone, Ph.D., Principal Scientist, Cell Line Development, MacroGenics, Inc.
12:15
Lunch on Your Own
1:45
Chairperson's Remarks
Andrew Snowden, Ph.D., Senior Scientist, Cell Culture Development, Biogen Idec
Andrew Snowden, Ph.D., Senior Scientist, Cell Culture Development, Biogen Idec
Developing Cell Lines for the Expression of Novel Molecules
Over the last 5 years, the generation of production cell lines expressing monoclonal antibodies has become increasingly routine, with platformized cell line development systems increasingly used. However with novel therapeutic candidates such as bispecific antibodies, or those that require enhanced specific activities such as ADCC, standardized cell line development platforms may no longer be applicable. This session will address the challenges of expressing these next generation therapeutics and strategies to successfully generate a suitable production cell line.
2:00
Avian Embryonic Stem Cells for the Industrial Manufacture of Vaccines and Antibodies
EB66® duck embryonic stem cells have been generated for the industrial production of vaccines and antibodies with enhanced ADCC. Such cells maintain the desirable features of ES cells and display attractive industrial characteristics. Fully controlled cell banks are available and a biologic master file was filed with the FDA. EB66® cells have already been licensed to over 28 Biotech and Pharma companies worldwide.
Majid Mehtali, Ph.D., Managing Director and Chief Scientific Officer, Vivalis, France
EB66® duck embryonic stem cells have been generated for the industrial production of vaccines and antibodies with enhanced ADCC. Such cells maintain the desirable features of ES cells and display attractive industrial characteristics. Fully controlled cell banks are available and a biologic master file was filed with the FDA. EB66® cells have already been licensed to over 28 Biotech and Pharma companies worldwide.
Majid Mehtali, Ph.D., Managing Director and Chief Scientific Officer, Vivalis, France
2:30
Case
Study
Scalable Production of Novel Bispecific IgG-Like AntibodiesStudy
Bispecific antibodies (BsAbs) represent a new class of potent therapeutics. However, production of BsAbs is frequently hindered by biophysical instability. We have expressed genes encoding IgG-like BaAbs with engineered stable single-chain Fv domains, in CHO cells. We will present our experience in generating cell lines capable of scaleable production of BsAbs that exhibit attributes similar to traditional monoclonal antibodies.
Gisela Chiang, Ph.D., Principal Scientist, Clinical Cellular Engineering, Biogen Idec
3:00
Networking Refreshment Break
Overcoming the Barriers to Using New Expression Systems - Mammalian and Non-Mammalian
Increasingly in the biotherapeutics industry, derivatives of the Chinese hamster ovary (CHO) cell line have become the host cell line of choice for the production of recombinant proteins. However, a number of interesting alternative expression systems, based on alternative host lines from both mammalian and non-mammalian origin, are now being developed or are already in use. These systems and cell lines may exhibit significant advantages in terms of titer or specific product quality attributes (e.g. Glycan) to CHO. The talks in this session will focus on these new systems, their advantages and how to overcome any potential hurdles and barriers to their use.
3:30
Expression of Recombinant Proteins using the PER.C6 Human Cells
In the past few years, PER.C6® cells have gained a lot of attention primarily due to the very high expression levels of monoclonal antibodies. Recently, we have successfully used this cell line to express various recombinant proteins at high levels and with the desired quality characteristics. This presentation will describe our experiences in expressing and characterizing these recombinant proteins using the PER.C6® human cells.
John H. Chon, Ph.D., Senior Director, BioProcess R&D, Percivia, LLC
In the past few years, PER.C6® cells have gained a lot of attention primarily due to the very high expression levels of monoclonal antibodies. Recently, we have successfully used this cell line to express various recombinant proteins at high levels and with the desired quality characteristics. This presentation will describe our experiences in expressing and characterizing these recombinant proteins using the PER.C6® human cells.
John H. Chon, Ph.D., Senior Director, BioProcess R&D, Percivia, LLC
4:00
Bioproduction of Therapeutic Proteins in the Lemna Expression (LEX) System
Lemna, or duckweed, is a small aquatic plant that can be quickly transformed to produce recombinant proteins in a contained and controlled bioprocessing environment. The benefits of the LEX™ System include a high transformation efficiency, rapid clonal growth in a completely closed and aseptic environment using a simple and relatively inexpensive production format. To date, over 30 different human therapeutic proteins have been produced by the LEX System™, including several hard-to-make proteins and over 16 different monoclonal antibodies with the option of glycan optimization.
Lynn Dickey, Ph.D., Vice President, Research and Technology Development, Biolex Therapeutics
Lemna, or duckweed, is a small aquatic plant that can be quickly transformed to produce recombinant proteins in a contained and controlled bioprocessing environment. The benefits of the LEX™ System include a high transformation efficiency, rapid clonal growth in a completely closed and aseptic environment using a simple and relatively inexpensive production format. To date, over 30 different human therapeutic proteins have been produced by the LEX System™, including several hard-to-make proteins and over 16 different monoclonal antibodies with the option of glycan optimization.
Lynn Dickey, Ph.D., Vice President, Research and Technology Development, Biolex Therapeutics
4:30
Production of Biopharmaceuticals in Human Amniocytes
Human CAP (CEVEC's Amniocyte Production) cells allow for stable and high yield production of recombinant proteins, with excellent biologic activity and therapeutic efficacy, as a result of authentic posttranslational modification. Based on CAP cells a transient expression system has been developed, that enables extremely high production yields of recombinant proteins within a few days. Thus, CAP and CAP-T technologies offer the use of only one unique platform for early preclinical development through to clinical supply of recombinant biotherapeutics.
Hartmut Tintrup, Ph.D., Director, Marketing and Business Development, Cevec Pharmaceuticals GmbH, Germany
Human CAP (CEVEC's Amniocyte Production) cells allow for stable and high yield production of recombinant proteins, with excellent biologic activity and therapeutic efficacy, as a result of authentic posttranslational modification. Based on CAP cells a transient expression system has been developed, that enables extremely high production yields of recombinant proteins within a few days. Thus, CAP and CAP-T technologies offer the use of only one unique platform for early preclinical development through to clinical supply of recombinant biotherapeutics.
Hartmut Tintrup, Ph.D., Director, Marketing and Business Development, Cevec Pharmaceuticals GmbH, Germany
5:00
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