Cell Line Development and Engineering 
Event Information
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Agenda
| Day One Monday, June 23, 2008 | DAY ONE | DAY TWO | DAY THREE | | |
| 8:00 | Chairperson's Opening Remarks John Joly, Ph.D., Director of Early Stage Cell Culture, Process Development, Genentech, Inc. |
| Timelines and Trade-Offs - Finding the Balance between Speed, Quality and Productivity | |
| 8:15 | Approaches to Enable Short Stable Cell Line Development Timeline without Reducing Productivity Monoclonal antibodies are an important class of therapeutic proteins, which often require high titer production processes. Therefore, rapid generation of stable antibody-producing CHO cell lines with high productivity is critical. To achieve a short timeline without compromising productivity, one approach we explored is to simultaneously transfect two plasmids into CHO cells for stable antibody cell line generation. We call this approach double selection. Another approach we explored is to adapt the host cells to grow to high cell density. This presentation will discuss the platform employed at Genentech to generate stable antibody-producing cell lines, in conjunction with these new technologies. Amy Shen, Scientist, Early Stage Cell Culture, Genentech, Inc. |
| 8:45 | Case Study on Streamlining Mammalian Cell Line Development Processes while Simultaneously Shortening Timelines Pfizer has focused on streamlining our cell line development strategies to rapidly bring protein therapeutics to market. This presentation will describe the sequential prosecution of an IgG molecule through two different mammalian expression systems incorporating improvements in screening, feed strategies and the establishment of a serum-free process. Sybille S. Galosy, Ph.D., Senior Principal Scientist, Cell Line Development, Global Biologics, Pfizer Inc |
| 9:15 | BI HEX® - A Holistic Concept for Fast Time to Clinic Most biopharmaceuticals are currently expressed in Chinese hamster ovary (CHO) cells. Process development faces at least two challenges: (i) the need to achieve high product titer and (ii) short development times to obtain clinical grade material. Boehringer Ingelheim's CHO high expression BI HEX® cell line generation concept combines many improvements. These include unique genetic elements, HTS concepts to reliably obtain highly productive clones that grow to high density in serum-free chemically defined media with productivities > 50 pg/cell/d and product titers of > 6g/L in a fed-batch process for mAbs. Torsten W. Schulz, Ph.D., Associate Director, Biopharmaceutical Process Science, Boehringer Ingelheim Pharma GmbH & Co. KG, Germany |
| 9:45 | Poster/Exhibit Viewing and Refreshment Break |
| 10:15 | Improving the Accuracy, Speed and Reproducibility of Titer Determination for the Clone Selection Process Accurate titer determination remains a challenge, for cell line development, with less than desirable precision, accuracy, and throughput. A new technology was evaluated that provides a > 10x decrease in hands-on time and turnaround time vs. both HPLC and ELISA while providing accuracy and precision comparable to that of Protein A HPLC without the HPLC waste or maintenance. Keith A. Davis, Ph.D., Scientist, WPGRD, Global Biologics, Pfizer Inc |
| 10:45 | Rapid Generation, Screening and Development of Highly Productive CHO Cell Lines We have incorporated a series of processes to both enhance the ability to isolate high producing clones and also decrease the time in screening and development to facilitate "transfection to MCB" in 8-9 months. We evaluate multiple candidate IgG-molecules in each project. Our cell line development paradigm includes FACS cloning, 2-3 levels of miniaturization screening, phenotypic stability studies, molecule quality evaluation, adventitious agents testing, bioreactor studies and in some cases TOX bioreactor campaigns. By mapping and clearly understanding the cell line generation/development processes, precise timelines are generated that enable groups to work closely together both within projects and in paralleled projects. Christopher Hatfield, Assistant Senior Biologist, BioProcess R&D, Eli Lilly and Company |
| 11:15 | Accelerating Development of Recombinant Antibody Therapeutics: Strategies for Early and Clinical Supply from CHO Cells Abstract to come. Diane Hatton, Ph.D., Head of Cell Engineering, Development, MedImmune Limited, United Kingdom |
| Technology Workshop | |
| 11:45 |  Cell Line Development Using a PER.C6® Host Cell Line - A Highly Efficient Expression PlatformThe PER.C6 cell line was developed from human primary retina cells, and exhibits favorable characteristics for the production of recombinant proteins. A serum-free, suspension adapted PER.C6 cell line is used for the expression of recombinant human proteins. In this presentation, data on IgGs will be discussed. Using commercially available expression vectors and standard selection and cloning methodologies, cell lines with high cell specific productivities of over 50 pcds were generated without the need for amplification. In addition to high specific productivity, the extreme cell densities achieved in culture yielded over 5.8 g/L in fed-batch and over 13 g/L in XD™ processes. Gene W. Lee, Director of Cell Line Development, Percivia |
| 12:15 | Lunch on Your Own |
| 1:40 | Chairperson's Remarks Dennis M. Kraichely, Ph.D., Principal Research Scientist, Expression Technologies, Centocor, Inc. |
| From Transfection through Clone Selection | |
| 1:45 | Approaches to Improve Cell Line Development There are many opportunities to improve the speed, efficiency, and outcome of mammalian cell line development programs. We have focused our efforts at improving the efficiency of generating CHO cell lines antibodies in several areas. Information will be presented on host cell improvement, vector modification, enrichment of high-expressing cell lines, high-throughput screening, and scaled-down modeling. Nels Pederson, Ph.D., Associate Director, Cellular Engineering, Biogen Idec |
| 2:15 | Novel Approach for Cell Line Development - The Development of a Fully-Automated Workcell for Shake Flask Cell Culture Shake flask cultivation is routinely used to rank and select cell lines prior to bioreactor evaluation. Shake flask cultivation requires repetitive and laborious manual manipulations that includes decapping/recapping flasks, cell counting, subculturing, feeding and data recording. We have developed a novel "Workcell" robot to automate the aforementioned manipulations. The development and functions of the Workcell will be presented. Lin Zhang, Associate Research Fellow, Global Biologics, Worldwide Pharmaceutical Sciences, Pfizer Inc |
| 2:45 | Clone Selection Using ClonePix: Benefits and Limitations Clone selection is a key step in cell line development. Recent advances have focused mainly on high throughput instrumentation that allows more clones with desired phenotypic attributes to be screened, thereby enhancing the ability to find the best producers. This talk will focus on our experience using the ClonePixFL instrument, highlighting the development of better clone selection algorithms and the need to develop appropriate cloning media. Peggy Lio, Process Sciences Fellow, Invitrogen |
| 3:15 | Using EESYR Cell Lines to Facilitate Production of VelocImmune Antibodies We have developed a clinically enabling CHO cell line capable of high titer antibody production, as well as methods for resource-efficient high-throughput cell line construction. Antibody genes are integrated into a defined genomic locus (EESYR) by site-specific recombination. Within 6 weeks of transfection, EESYR cells expressing antibodies consistently yield bioreactor titers greater than 1 gram per liter. Robert Babb, Ph.D., Senior Staff Scientist, Regeneron Pharmaceuticals, Inc. |
| 3:45 | Poster/Exhibit Viewing and Refreshment Break |
| Afternoon Plenary Session | |
| Keynote Address | |
| 4:15 | Genomic and Proteomic Tools to Elucidate How Mammalian Cell Factories Achieve High Level Productivity The advent of systems biology has created a new paradigm for understanding the dynamic genetic/regulatory and metabolic networks that determine the functional competence of mammalian cell factories in vitro. How can we access and utilise genome-scale information to reduce the time taken to generate cell lines which combine a variety of desirable multigenic traits; high cell specific productivity, correct product processing and extended survival in the in vitro environment? David James, Ph.D., Professor of Bioprocess Engineering, Department of Chemical and Process Engineering, University of Sheffield, United Kingdom |
| Keynote Address | |
| 5:00 | How do ER Stress and Oxidative Stress Impact Protein Production? The endoplasmic reticulum (ER) is the site where proteins destined for secretion fold into their appropriate three-dimensional conformation. Protein folding in the oxidizing environment of the ER is energy dependent is coupled with disulfide bond formation. Recent studies demonstrate that protein folding in the ER can cause oxidative stress that reduces the efficiency of protein folding. Strategies will be presented that improve the redox status of the ER and improve protein secretion. Randal J. Kaufmann, Ph.D., Warner-Lambert/Parke-Davis Distinguished Professor of Biological Chemistry and Internal Medicine, Howard Hughes Medical Institute, University of Michigan Medical Center |
| 5:45 | Exhibit Hall Networking |
| Day Two Tuesday, June 24, 2008 | DAY ONE | DAY TWO | DAY THREE | | |
| 8:00 | Chairperson's Opening Remarks John Mott, Ph.D., Director, Bioprocess R&D, Cell Line Development, Pfizer Global Biologics |
| From Transfection through Clone Selection (continued) | |
| 8:15 | Rapid Generation of High Productive Cell Lines using the Selexis Expression System Over the past two years Merrimack Pharmaceuticals has successfully developed and implemented a process for rapid generation of highly productive cGMP cell lines. This process is based on the use of the Selexis™ expression system. In this talk, we will describe our process and the results achieved with several therapeutic antibodies and proteins. Jose Varghese, Director, Process Development, Merrimack Pharmaceuticals |
| 8:45 | Predictive Modeling: Lessons Learned from Analysis of Early Clone Secretion Heterogeneity One of the largest bottlenecks in clone selection is currently scale-up and characterization of clones to identify high producers. In search of an early predictor of final productivity, we tracked the secretion heterogeneity of clones from the 6-well stage up to shake flask. We are currently analyzing the rank order of clones to determine whether early secretion heterogeneity can be used to predict later shake flask productivity. Genova Richardson, Senior Research and Development Scientist, Cell Sciences and Development, SAFC Biosciences |
| 9:15 | Site-Specific Integration and Gene Disruption Zinc Finger Nucleases (ZFNs) represent a new class of tools for genome engineering. Here we show that ZFNs promote high-efficiency site-specific insertion and targeted gene deletion across a variety of transformed and primary cell types. Application of these novel genetic tools in cell line development and cell therapy will be discussed. Philip D. Gregory, Ph.D., Vice President, Research, Sangamo BioSciences Inc. |
| 9:45 | Poster/Exhibit Viewing and Refreshment Break |
| 10:15 | Clone Selection in Early Stage Process Development of Monoclonal Antibody Therapeutics Recombinant protein therapeutics is routinely produced in mammalian host cell lines. The need for rapid entry of new molecular entities into clinical development requires rapid process development using productive cell lines. The first step in this process is usually the selection of a clone that exhibits high productivity, acceptable metabolite profiles while maintaining product quality attributes. This presentation will highlight our strategy for clone selection in the context of platform processes and aggressive timelines. Ashraf Amanullah, Ph.D., Principal Engineer and Group Leader, Genentech, Inc. |
| 10:45 | Key Issues Facing Cell Line Developers and Cell Culture Engineers Cell line development and cell culture process development are complex and interdependent. Decreasing timelines have placed pressure on both of these groups that traditionally perform their functions in isolation to work in parallel. Some of the challenges and unexpected benefits of this change in the industry will be discussed. Laurie Donahue-Hjelle, Ph.D., Director of Cell Line Development, Invitrogen Corporation |
| Technology Workshop | |
| 11:15 |  Cell Line Characterization and Culture Media Optimization with Phenotype MicroArraysPhenotype MicroArrays are a new process development tool for cell-line characterization and high-throughput testing of cell culture conditions that affect metabolism, growth, or productivity. Every cell line yields a unique fingerprint that can be used to monitor the phenotypic effects of media changes, validate cell line stability and look for factors that may help predict bioreactor productivity. Ben Bowen, Ph.D., Vice President of Business Development, Biolog |
| 11:45 | Luncheon and Poster/Exhibit Viewing |
| 1:10 | Chairperson's Remarks Laurie Donahue-Hjelle, Ph.D., Director of Cell Line Development, Invitrogen Corporation |
| Improving Quality - Overcoming Unexpected Product Quality Characteristics and Utilizing Techniques for Selecting Better Clones | |
| Featured Presentation | |
| 1:15 | Glycosylation and Protein Engineering: Effect on Activity, Stability and Functionality It is established that glycosylation can have a positive, negative or, seemingly, neutral impact on the in vivo activities of therapeutic glycoproteins. Production of homogeneous natural glycoforms, optimal for a given function, is now possible and has the benefit of not being immunogenic. However, the impact on function(s) is restricted. Protein engineering has the potential to modulate multiple functions but introduces a potential for immunogenicity. There is a danger of focusing on individual functions, at the expense of a holistic view, e.g. antibody engineering to improve pharmacokinetics can impact interactions of IgG-Fc with virus induced IgG-Fc receptors. Roy Jefferis, Ph.D., Professor of Molecular Immunology, Division of Immunity & Infection, University of Birmingham, United Kingdom |
| 1:45 | Effects of Cell Culture Conditions on Glycosylation The glycosylation of recombinant proteins secreted by mammalian cells is essential for full biological activity in vivo. However, the glycan structures attached to proteins are highly variable. Critical parameters are associated with the cell line, the culture media and the specific protein synthesized. These effects will be discussed in the context of cell culture bioprocesses where consistency of glycosylation profiles is essential. Michael Butler, Ph.D., Professor of Microbiology, University of Manitoba, Canada |
| 2:15 | Advances and Challenges in Post-Translational Modifications of Biopharmaceuticals Currently, mammalian cells are the dominant production system for antibodies and most other therapeutic proteins because they can perform complex post-translational modifications that are often required for efficient secretion, drug efficacy and stability. This presentation will provide examples of analytical and preventative advances in protein modifications such as methionine oxidation, asparagine deamidation, misfolding & aggregation. Nigel Jenkins, Professor, Principal Investigator, National Institute for Bioprocessing Research and Training, University College Dublin, Ireland |
| 2:45 | Are Variable Results Utilizing Gene Copy Number Assays an Indication of Genetic Instability? Cell substrates currently used for recombinant DNA products require testing for genetic stability as part of cell line characterization. The maintenance of copy number serves as a useful marker, signaling genetic changes in the host/vector system, from cell bank to production. Quantitative PCR of genomic DNA can be used to detect specific recombinant genes within the cell line of interest. A bacterial model host/vector system was used to evaluate variability introduced at various steps in the copy number analysis. Even with a high degree of intra-assay accuracy and precision, the quantitative data acquired from this sensitive assay suggests that an acceptable range of variability is not an indication of genetic instability. Deborah Lee Dormady Letham, Ph.D., Scientist II, Methods Development, Biopharmaceutical Services, Charles River Laboratories |
| 3:15 | Poster/Exhibit Viewing and Refreshment Break |
| 3:45 | Transcriptomic and Proteomic Analysis of NS0 Cells Gene and protein expression profiling has been established to gain deeper insights into the intracellular response of antibody producing NS0 cell lines to increasing cell density in perfusion cultures and to provide a detailed understanding of the complex cellular functions and mechanisms that constraints survival, growth and productivity, thus allowing the rational modifications of metabolic pathways to improve cell line performance. The gene expression profiles in the changing environment of perfusion culture were compared to the profiles generated from chemostat cultures where a controlled and defined steady state condition permitted the analysis of cell response to anti-apoptotic signals. Britta Krampe, Ph.D. Student, Researcher, School of Chemical and Bioprocess Engineering, University College Dublin, Ireland |
| 4:15 | Sensitive Mass Spectrometric Methods to Detect Sequence Variants Caused by Mutations in Production Cell Lines Abstract to come. Viswanatham Katta, Senior Scientist, Protein Analytical Chemistry, Genentech, Inc. |
| 4:45 | Immunogenicity of Antibodies Clinical studies have highlighted that one of the major problems associated with the treatment of disease with therapeutic antibodies is the development of an anti-therapeutic protein immune response. Such responses ranging from 30-100% have been observed with antibody treatments for prostate cancer and adenocarcinoma patients using anti-PSMA and anti-CEA-CPG2 conjugates, respectively. More recently the use of bi-specific antibodies has also resulted in high incidence of anti-therapeutic immunogenicity in the treatment of metastatic breast cancer (anti-HER2/CD3) and Hodgkin's disease (anti-CD16/CD30). T cell epitopes present in the sequence of antibody therapeutics have been targeted in order to overcome problems associated with immunogenicity against antibody therapeutics. To this end two new technologies have been developed that allow the accurate identification of T cell epitopes and selection of lead biologics with reduced immunogenicity. These platforms have been applied to the generation of novel anti-cancer antibody therapeutics resulting in molecules with a lower risk of immunogenicity. Frank Carr, Ph.D., Director of Biologics Research, Antitope Ltd, United Kingdom |
| 5:15 | Close of Day Two |
| Day Three Wednesday, June 25, 2008 | DAY ONE | DAY TWO | DAY THREE | | |
| 8:00 | Chairperson's Opening Remarks Kevin J. Kayser, Ph.D., Research and Development Manager, Cell Line Engineering, SAFC Biosciences |
| Advances in Cell Line Knowledge and Understanding | |
| 8:15 | Stability/Instability of Recombinant Protein Expression in Engineered Mammalian Cell Lines Stability of expression of recombinant protein throughout the timescale of cell line expansion and process scale-up remains a key priority for effective bioprocessing. I will describe our experience of assessment of stability/instability in recombinant mammalian cell lines at the molecular level, focusing on gene localization and DNA methylation analysis, and review approaches that may predict or favor stability. Alan J. Dickson, Ph.D., Professor of Biotechnology, University of Manchester, United Kingdom |
| 8:45 | Differential Expression Profiling of Industrially Relevant CHO Cell Phenotypes Using a Proprietary CHO-Specific Microarray and Proteomics Technology Platforms Abstract to come. Mark Melville, Ph.D., Principal Research Scientist I, Drug Substance Development, Wyeth BioPharma |
| 9:15 | Use of Microarrays and Proteomics to Find Biomarkers in CHO NICB, in collaboration with Wyeth Biopharma (Andover, USA and Grangecastle, Ireland), using a proprietary CHO WyeHamster2a microarray chip, 2D-DIGE and Mass Spectrometry, have carried out expression profiling analyses of multiple production cell line samples, representing a number of phenotypic categories, including high cell growth rate, high cell density, high cell viability, cellular productivity (Qp), as well as low lactate and ammonia production. Paula Meleady, Ph.D., Program Leader, Proteomics Core Facility, National Institute for Cellular Biotechnology, Dublin City University, Ireland |
| 9:45 | Apoptotic and Autophagic Death in CHO Cell Culture: Its Prevention through Cell Engineering Recent studies show that CHO cells, upon nutrient deprivation, are subjected to two types of programmed cell death (PCD), apoptosis and autophagy. Therefore, we revisit the issue of cell death in CHO cell culture with the concept of autophagy in mind, in order to achieve a maximum production of foreign proteins by protecting cells from both types of PCD. Gyun Min Lee, Professor, Biological Sciences, KAIST, Korea |
| 10:15 | Poster/Exhibit Viewing and Refreshment Break |
| 10:45 | Metabolomics and its Applications to Improve Bioprocessing Unbiased global metabolic profiling is an approach for obtaining a "snapshot" of the metabolism of a population of cells in a bioreactor. As such, it has had particular utility in the area of upstream bioprocess optimization of biopharmaceuticals. The hundreds of metabolites detected by global profiling offer a vastly expanded panel of biochemicals for monitoring media formulations for unexpected nutrient depletion or toxicant accumulation. Further, this panel has yielded detailed insight into why such metabolites diminish or accumulate. Finally, metabolites that directly impact key areas of phenotypic output such as stability of the transgene, glycosylation, and cell viability are often identified by distinct media formulations by metabolomics. Michael Milburn, Ph.D., Chief Scientific Officer, Metabolon |
| 11:15 | Overview of RNAi Application in Cell Line Development This overview summarizes recent advances in RNAi technology applications for cell line development and engineering, including 1) target gene discovery and validation; 2) siRNA stable cell line generation; 3) improvement of cell line productivity; 4) target gene silencing in suspension cell culture; and 5) other potential applications. Jianguo Yang, Ph.D., Scientist and Group Leader, Cell Line Development Group Process Cell Culture and Fermentation, MedImmune, Inc. |
| 11:45 | Multiple Gene Knockout in Mammalian Cells Using Zinc Finger Nucleases: Characterization of a DHFR-/-GS-/-FUT8-/- Triple Knockout CHO Cell Line Rational genome engineering in mammalian cells is of enormous potential across basic research, bioprocess, drug-discovery, as well as cell-based medicines. To this end we have developed a technology that enables rapid genome editing via the application of designed zinc finger protein nucleases (ZFNs). We will showcase the application of this approach to custom cell engineering through characterization of a triple gene knockout cell line. Trevor Collingwood, Team Leader, Enabling Technologies, Sangamo BioSciences Inc. |
| Technology Workshop | |
| 12:15 |  PACE Cell TechnologyThe RP Shift® process increases biopharmaceutical yields from 3 to 7-fold in commercial cellular systems by prolonging the lifetime of cells in culture and increasing synthesis and secretion rates of protein. The PACE™ system couples RP Shift® pre-engineered cells with unique expression vectors to maximize titers and minimize process development times. Thomas Primiano, Ph.D., President & CEO, CDI Bioscience |
| 12:45 | Luncheon and Poster/Exhibit Viewing |
| 2:00 | Chairperson's Remarks Morris Rosenberg, D.Sc., Senior Vice President, Development, Seattle Genetics |
| Will Rodent Cell Production Go the way of the Dinosaur or be Eclipsed by Better Systems? | |
| 2:05 | Economics of Protein Production by Transgenic Animals and Cell Culture - Choosing One Production of therapeutic proteins with human glycosylation patterns has pushed manufacturers to utilize mammalian cell culture as a preferred expression system. However, glycosylation is still not identical to the patterns found in naturally produced human proteins and glycosylation can shift with environmental conditions. High manufacturing costs prompted the development of alternative transgenic production systems. This presentation will discuss the history and current state of transgenic goat production systems and compare its economics with the cost of mammalian cell culture. Richard Schoenfeld, Ph.D., Principal, BioWorks Consulting |
| 2:35 | Stable and High Volumetric Production of Glycosylated Human Recombinant IFN 2b in HEK293 Cells We have engineered a HEK293 cell line that produces up to 315 mg of human recombinant IFN 2b per litre of serum-free medium within 7 days and developed a robust and efficient process yielding 98% pure IFN 2b with greater than 70 % recovery. Purified IFN 2b was glycosylated and highly sialylated as shown by mass spectroscopy analysis. Perfusion culture in a 3L bioreactor showed stable IFN 2b production over 35 days. These results demonstrate that the HEK293 cell line is an efficient host for the large-scale production of glycosylated human IFN 2b and potentially other recombinant proteins. Yves Durocher, Ph.D., Senior Research Officer, Animal Cell Culture Technology, Bioprocess Sector, Biotechnology Research Institute, National Research Council of Canada |
| 3:05 | Networking Refreshment Break |
| 3:30 | Production of Fully Functional Monoclonal Antibodies by Glycoengineered Yeast Strains at Over 1 g/L All currently approved monoclonal antibodies are produced in mammalian cells with N-linked glycan structures similar but not identical to their human equivalents. GlycoFi has established a glycoengineered yeast strain capable of producing greater than 1g/L of a fully assembled and functional monoclonal antibody in a readily scaleable process with highly uniform human N-linked glycans. This paper will discuss GlycoFi / Merck's ability to produce fully functional therapeutic monoclonal antibodies at commercially viable expression levels. Stefan Wildt, Ph.D., Director, GlycoFi, Merck & Co., Inc. |
| 4:00 | Metabolic Engineering Using the CHEF1 Expression System The CHEF1 expression system was developed for protein expression in CHO cells. A unique feature of the CHEF1 system is that it allows for multiple transfections with different genes enabling the co-expression of multiple proteins. Thus, the system is ideal for using metabolic engineering to improve the expression and activity levels of therapeutic proteins. An example of using the CHEF1 system to perform metabolic engineering will be discussed. Andy Walker, Ph.D., Staff Scientist, Cell Culture Process Development, CMC, ICOS Biologics |
| 4:30 | Efficient Production of Therapeutic Proteins in the LEX System™ Lemna 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 SystemSM include high transformation efficiency, rapid clonal growth with stable high expression using a simple and inexpensive production format. Lemna provides an ideal platform for expression of a broad range of protein products including cytokines, monoclonal antibodies, and vaccines. To date, over 34 different human therapeutic proteins have been produced by the LEX System™. Kevin Cox, MS, Head of Genetic Engineering, Biolex Therapeutics |
| 5:00 | Close of Conference |
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