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Optimizing Mammalian Cell Lines - Day 1


Optimizing Mammalian Cell Lines 

Gene expression in mammalian cells is the foundation for biopharmaceutical production. As more protein-based products head into development, the need to refine processes for optimizing cell line development increases. Reducing the time needed to identify high-expressing clones that produce protein with desired quality is essential for trimming a project's overall costs and reducing program risk.

The Optimizing Mammalian Cell Lines meeting features experts sharing their protocols and case studies illustrating the steps they have taken to reach enhanced expression. Their insights and experiences will provide real-world details to help you conquer the challenges of cell line development. As you seek to enhance your skills and knowledge, make time to join the information exchange and networking with colleagues from around the world who share common cellular goals on the path to protein production.


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Wednesday, August 24

7:30 am Registration & Morning Coffee

 

CELL LINE DEVELOPMENT 

8:25 Chairperson's Remarks

Susan Sharfstein, Ph.D., Associate Professor, Nanobioscience, College of Nanoscale Science and Engineering, University at Albany

8:30 Opening Keynote Presentation:
Cell Engineering in a Genomics Era

Michael BetenbaughMichael Betenbaugh, Ph.D., Professor, Chemical & Biomolecular Engineering, Whiting School of Engineering, Johns Hopkins University - Biography 

The advent of the Chinese Hamster Genome will transform the ways in which cell and metabolic engineering occur in the coming decades.  We will now have access to much greater amounts of genomic, proteomic, metabolomics, and glycomics data.  How this information is used to alter cell behavior by genetic manipulation or environmental changes will shape mammalian biotechnology in the coming decades.  In this presentation, we will outline several ways in which we are utilizing genomic data to alter mammalian cell performance.  In one case study, we are evaluating the role of microRNAs in the activation of the apoptosis cascade.  Chinese hamster ovary (CHO) cells were induced to undergo apoptosis by exposing the cells to nutrient-depleted media. A microarray comparison of known microRNA's in CHO cells exposed to fresh or depleted media revealed up-regulation of the mouse miR-297-669 cluster in CHO cells, including the specific microRNA Mmu-miR-466h. We further hypothesized that up-regulated mmu-miR-466h inhibits anti-apoptotic genes and induces apoptosis and a combination of bioinformatics and experimental tools predicted 38 anti-apoptotic targets.  Several genes were selected from this anti-apoptotic subset based on nucleotide pairing complimentarity.  The inhibition of the mmu-miR-466h lowered the expression levels of these genes, increased cell viability, and decreased apoptosis activity. In separate studies we are applying proteomic tools to CHO cells and evaluating what proteins may be critical to optimizing bioprocessing. 

9:00 Featured Presentation:
Novel Vector Technologies for Enhanced CHO Cell Line Development

Thomas JostockThomas Jostock, Ph.D., Technology Network Leader, Integrated Biologics Profiling, Novartis Pharma AG - Biography 

The current Novartis CHO platform allows generation of high yielding production cell lines with short cycle times. Our strategy for further optimising speed and yield of the platform combines internal efforts with systematical screening and evaluation of external know-how. By integrating internal and external technologies, we are aiming for further reducing cycle times and screening efforts of cell line development. Some novel vector technologies that we have evaluated to improve our platform towards high yielding fast processes, including a new selection marker and a targeted integration technology, will be presented. 

9:30 Early Markers of Production Instability of Recombinant Chinese Hamster Ovary Cell Lines

Ulrich GoepfertUlrich Göpfert, Ph.D., Senior Research Scientist, Pharma Research and Early Development (pRED), Biologics Research, Roche Diagnostics GmbH - Biography 

Production instability of manufacturing cell lines can be associated with methylation and silencing of the heterologous promoter. We have established methylation-specific real-time qPCR for the rapid and sensitive measurement of hCMV-MIE methylation in multiple cell lines and provide evidence that hCMV-MIE methylation and transgene copy numbers can be used as early markers to predict production instability of recombinant CHO cell lines. These markers should provide the opportunity to enrich stable producers early in cell line development and allow developers to put more emphasis on other criteria, such as product quality and bioprocess robustness.
 

10:00 Networking Coffee Break with Exhibit and Poster Viewing

10:45 Development of a High Yielding, Scalable CHO Transient Expression System

Gary Pettman, Team Leader, Early Expression and Supply, MedImmune

A rapid and productive expression strategy is required to meet the increasing requirements for research grade material during early drug development. The development, optimization and implementation of a scalable, high yielding proprietary CHO transient expression system will be presented. The system has been successfully scaled up to 250L and is capable of expressing several hundred mg/L of recombinant protein and is used routinely to provide multi-gramme quantities of pre-clinical grade material for Biopharmaceutical development.

11:15 Development of Predictive Methods for Cell Line Selection and Process Development

Arnaud PerilleuxArnaud Perilleux, Scientist, Upstream Processing, Biotech Process Sciences, Merck Serono SA - Biography 

This study focuses on the development of a robust fed-batch platform process in a 96-deep well plate system for cell line screening and process development. The system is aimed at screening hundreds of cell lines in suspension with a feeding system and a run duration which are in line with the platform bioreactor process used at Merck Serono. The cell line screening system demonstrated cell line specific culture performances which are in good agreement with those of cultures carried out at larger scales (spin tubes and lab-scale bioreactor cultures) and are predictive in terms of final titer. Challenges such as scale alignment, robustness of the 96-deep well plate system, integration of robotic liquid handling systems and high throughput analytics will be discussed. Additionally, the 96-deep well plate cultivation system can be efficiently used to complement existing development tools with increased throughput and enhanced potential for Design of Experiment (DoE) approaches. This new method was implemented to develop media and feeds for next generation upstream platform processes.

Sponsored by
Pall_LifeSciences 
11:45 Micro-24 Microbioreactor: "High-Throughput" Bioreactors Redefining Cell Line Optimization and Process Development…Today
Tiffany D. Rau, Ph.D., Global Technology & Technical Manager, Pall Corporation 
New tools and methods are available to screen and develop processes under controlled conditions earlier than ever before to allow ones organization to deliver a robust cell line and process earlier and also generate data for Quality by Design (QbD) initiatives. One tool that compliments QbD and delivering a robust platform for manufacturing is the Micro-24 Microbioreactor which is a 24 well reactor system where pH, dissolved oxygen and temperature are monitored and controlled.
 

12:00 pm Sponsored Presentation (Opportunity Available)

12:15 Luncheon Presentation (Sponsorship Opportunity Available) or Lunch on Your Own

 

CELL LINE SELECTION 

1:55 Chairperson's Remarks

S. Patrick Walton, Ph.D., Associate Professor, Chemical Engineering & Materials Science, Michigan State University

2:00 Optimal Selection of Cell Lines Producing Biopharmaceutical Human IgGs

Jolanda GerritsenJolanda Gerritsen, Ph.D., Technology Expert, Cell Line Development, Genmab B.V. - Biography 

Timelines and productivity are of the utmost importance in generating monoclonal antibody producing CHO cell lines. Therefore, we first implemented high-throughput automated picking of high producing clones both after transfection and sub-cloning using ClonePixFL (Genetix). Secondly, we employed miniaturized cell culture using sensor dish reader and μ-24 reactor systems (Applikon). These modifications reduced timelines as they require less expansion work due to their smaller scale. Furthermore, it considerably increased throughput in terms of the number of cell lines that can be screened, which as an additional advantage, can be grown under controlled fed-batch conditions, closely mimicking those in a bioreactor. As a result, the most optimal production cell lines for large-scale manufacturing are selected with faster timelines. This presentation highlights our novel high throughput and miniaturized cell line development process and provides insight into these specific improvements regarding time lines and product yield.

2:30 Applying Quality by Design Principles to Candidate Selection and Cell Line Development

Susan Dana JonesSusan Dana Jones, Ph.D., Vice President and Senior Consultant, BioProcess Technology Consultants - Biography 

Interweaving production cell line development with final candidate selection in an application of QbD is a further approach to reducing overall development timelines and insuring product quality. Such early product assessments demand earlier development of robust and reliable analytical methods to insure proper and timely results that can inform the decision making needed to select the lead candidate product from among the many that are under consideration and then to identify the cell line that produces the highest quality product. This talk will show how the integration of candidate selection, early analytical development, and cell line development can lead to more effective products that can be produced at lower cost.

3:00 Utilization of Non-AUG Initiation Codons in a Flow Cytometry-Based Method for Efficient Selection of Recombinant Cell Lines

Christine DeMariaChristine DeMaria, Ph.D., Senior Scientist, Therapeutic Protein Expression, Genzyme Corp. - Biography 

This high-throughput clone selection method utilizes a cell surface reporter to predict the expression level of any recombinant therapeutic protein. The reporter, which lacks a native translation initiation codon, is placed in the 5' UTR of the therapeutic so that both open reading frames are translated from the same mRNA. Flow cytometry sorting of uncloned pools is used to isolate high producing cell lines based on reporter expression. Subsequent flow cytometry screening during clone expansion enables resources to be focused solely on clones with both high and stable therapeutic expression.

3:30 Networking Refreshment Break with Exhibit and Poster Viewing

 

TRANSFECTION / ELECTROPORATION 

4:15 Flow-Through Electroporation Based on Constant Voltage for Large-Volume and Enhanced Cell Transfection

Chang LuChang Lu, Ph.D., Associate Professor, Chemical Engineering, Virginia Tech - Biography 

Here we present a novel flow-through electroporation method for delivery of DNA into cells with a processing rate up to ~20 ml/min based on disposable microfluidic chips, a syringe pump, and a low-cost direct current (DC) power supply that provides a constant voltage. Our technique eliminates the use of a pulse generator and the associated cost. More importantly, we show that by harnessing hydrodynamic effects of the flow, our technique enables the entire membrane surface to become uniformly permeabilized and this leads to greatly improved delivery and transfection. Our technique provides a drastically simple and effective approach to electroporation-based cell transfection.

4:45 Nanochannel Electroporation–Giving Cells a Shot

L. James LeeL. James Lee, Ph.D., Helen C. Kurtz Professor, Chemical and Biomolecular Engineering; Director, NSF Nanoscale Science and Engineering Center for Affordable Nanoengineering of Polymer Biomedical Devices (CANPBD), The Ohio State University - Biography 

We describe a new technology, nanochannel electroporation (NEP) allowing transfection of many small sized and delicate cells with precise control over dose and timing. Cell mortality from NEP is virtually zero. We show dose control effects on a variety of transfection agents such as oligonucleic acids, molecular beacon, quantum dots and efficient delivery of large plasmid DNA. Dosage controlled delivery to multiple cells is not achievable with any existing techniques.

 

5:15 Networking Reception, Last Chance for Poster and Exhibit Viewing

6:45 End of Day



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