Optimizing Protein Expression


EXPRESSION STREAM   May 11-12

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WEDNESDAY, MAY 11

7:00 am Registration and Morning Coffee

 

CHO & Mammalian Expression Systems 

8:30 Chairperson’s Opening Remarks

Shuangping Shi, Ph.D., Associate Principal Scientist, Bioprocess Development, Merck Research Lab, Merck & Co. 

 

» 8:40 Opening Keynote Presentation: 

Protein Expression in Drug Discovery – New Challenges, New Solutions 

Lorenz Mayr Lorenz M. Mayr, Ph.D., Executive Director, Unit Head Biology, Protease Platform, Novartis Pharma AG 

Success in drug discovery relies not only the appropriate selection of molecular targets, but also on the availability of high-quality recombinant protein and cell lines in sufficient amounts and on short time. Whereas protein expression has long been viewed as a mature science with no need for further improvement, current trends in drug discovery show an increased demand for fast & efficient production systems for recombinant proteins and protein complexes to cope with the demands for protein in sufficient amounts needed for modern hit discovery (HTS, FBS, structure) and lead optimization in discovery research. 

» 9:25 Featured Presentation: 

High-Level Recombinant Protein Production in CHO Cells Using Lentiviral Vectors and the Cumate Gene-Switch

Bernard Massie Massie, Ph.D., Director, Bioprocess Center, Institute of Research and Biotechnology, Research Council of Canada; President, l’Association de Thérapie Génique du Québec (ATGQ)

Fast and efficient production of recombinant proteins for structural and functional studies is a crucial issue for research and for industry. To this end, we have developed an efficient system to generate, in less than 6 weeks, pools of CHO cells stably expressing high-level of recombinant proteins (>100 mg/L). This system takes advantage of the efficient gene delivery of lentiviral vectors (LVs) in highly active transcription sites, coupled with the powerful cumate-regulated promoter that, not only allow for inducible gene expression, but is also 8-fold stronger in CHO cells than the optimized CMV5 promoter.
 

9:55 Transient Expression of an IL-23R Extracellular Domain Fc Fusion Protein in CHO vs. HEK Cells Results in Improved Plasma Exposure

John TraugerJohn Trauger, Ph.D., Group Leader, Genomics Institute of the Novartis Research Foundation

We found that the plasma exposure in mice of an IL-23R extracellular domain Fc fusion protein (IL23R-Fc) was improved about 30-fold when the protein was prepared by transient transfection of CHO vs. HEK cells. Characterization of the CHO- and HEK-expressed IL23R-Fc proteins indicated that the difference in their in vivo plasma exposure is due to differential glycosylation.

10:25 Coffee Break in the Exhibit Hall with Poster Viewing

 

USING microRNAs TO ENHANCE
PROTEIN EXPRESSION IN CHO
 

11:10 microRNAs: New Tools to Manipulate Protein Expression in CHO Cells

Niall Barron  Niall Barron, Ph.D., Program Leader, Mammalian Cell Engineering, National Institute for Cellular Biotechnology, Dublin City University

The ability of miRNAs to influence protein expression is now recognized as a fundamental layer of regulation within the cell. We will provide a brief overview of their biogenesis, genomic organization and mode of action, and then go on to describe some of the approaches we have taken to examine their potential application in the bioprocessing area, with particular emphasis on CHO cell engineering.

 

11:40 mRNA Stability and Antibody Production in CHO Cells: Improvement through Gene Optimization

Shuangping Shi Shuangping Shi, Ph.D., Associate Principal Scientist, Bioprocess Development, Merck Research Lab, Merck & Co.

Gene optimization substantially enhances antibody production in Chinese hamster ovary (CHO) cells. When gene optimization was applied to the heavy and light chain genes of a therapeutic antibody, we observed increased antibody production in transient transfection as well as in stable clones. It is also demonstrated that elevated heavy chain mRNA level was associated with the increase of antibody production. Further analysis suggests that the increased antibody expression is attributable to enhanced mRNA stability resulting from gene optimization.
 

     
Sponsored by
Icosagen
12:10 pm Luncheon Presentation I 
Fast, Simple and Efficient Method for Production of Biologics using Stable Extrachromosomal Expression System
Mart Ustav, Ph.D., Founder and CEO, Icosagen Cell Factory Ltd
Icosagen Cell Factory has developed efficient technological platform (QMCF Technology) for production of various recombinant proteins, antibodies and VLPs in mammalian (CHO or 293) cell system. Main goal of QMCF technology is stable maintenance and replication of appropriate expression plasmid, fast and feasible upscale and generation of production cell banks.

Sponsored byFujifilm Diosynth12:40 Luncheon Presentation II
Key Aspects of Managing Early Phase Development Programs for Long Term SuccessGeorge Koch, CSO, Fujifilm Diosynth BiotechnologiesHighlight several CMC best practices for preclinical activities. From selection of a cell line to release of the first clinical batch, product and process developers make decisions that have timeline, financial, and regulatory consequences.

1:10 Break 

 

Escherichia Coli & Clonal Cell Production 

1:30 Chairperson’s Remarks

Blaine Pfeifer, Ph.D., Assistant Professor, Chemical and Biological Engineering, Tufts University

1:35 Production of Antibody Mixtures and Bispecifics from Single Clonal Cells

John de Kruif, Ph.D., CSO, Merus Biopharmaceuticals BV

Pre-clinical and clinical studies demonstrate that mixtures of antibodies (mAbs) and bispecific mAbs represent next-generation biopharmaceuticals with improved specificity and efficacy. We have approached the manufacturing complexity of producing and developing these formats by using human mAbs that share the same identical germline-encoded light chain (‘single VL’). Transfection of cells with genetic constructs encoding 2 or 3 different ‘single VL’ mAbs results in the production of bispecific antibodies or mixtures of antibodies by clonal cells. Clonal cell lines show stable expression and high production levels for all mAb specificities even after > 60 passage doublings and show growth characteristics consistent with conventional mAb production cell lines. We show that native mass spectrometry-based analytical methods allow quantitative measurement of all antibody species in a complex mixture and that cation exchange chromatography can be used to efficiently separate bispecifics from the parental mAbs.  This technology facilitates the pharmaceutical production of next generation therapeutic antibodies based on intact IgG molecules.

2:05 Optimization of Protein Expression in E. coli: Best Practices and Unusual Tricks for the Production of Protein Suitable for Structural Studies

Rebecca PageRebecca Page, Ph.D., Assistant Professor, Biology and Principal Investigator, Molecular Biology & Cell Biology & Biochemistry, Brown University

I will be presenting both best practices and unusual (‘last ditch’) methods that are used to successfully express both prokaryotic and eukaryotic proteins in E. coli. Topics to be covered include: solubility tags, purification tags, chaperones and in vivo refolding, soluble expression through protein co-expression, and toxins and eukaryotic kinases.

    Sponsored byCaliper2:35  Automated Microfludic Analysis for Enhanced Optimization of Recombinant Protein Expression PlatformsMark Roskey, Ph.D., Senior Vice President, Applied Biology R&D, Caliper Life SciencesThis talk will focus on the use of high throughput  microfludics based electrophoretic  analysis for cell culture optimization and clone selection.  Applications discussed will include construct selection, factorial experiment design, analysis of antibody yield and purity, and glycan analysis.
 

3:05 Refreshment Break in the Exhibit Hall with Poster Viewing

3:50 Strategies for the Use of E.coli as an Expression Host for Challenging Proteins

Bingyuan WuBingyuan Wu, Ph.D., Research Scientist, Molecular & Protein Biosciences, Centocor R&D, Inc.

Escherichia coli has been a workhorse for recombinant protein expression due to its well-studied biology, fast growth, and high expression level. However, the expression of mammalian proteins in E. coli often turns out to be challenging. Here a few case studies will be presented on obtaining those difficult proteins using E. coli as an expression host.

4:20 The Challenges and Opportunities for Heterologous Reconstitution of Polyketide and Isoprenoid Natural Product Pathways through E.coli 

Blaine PfieferBlaine Pfeifer, Ph.D., Assistant Professor, Chemical and Biological Engineering, Tufts University

Polyketide and isoprenoid natural products display an impressive therapeutic range that has provided a strong motivation for new technologies to better access this medicinal potential. Equally motivating are the technical challenges associated with production processes reliant on the native host systems responsible for most polyketide and isoprenoid compounds. As a result, heterologous biosynthesis has gained noticeable traction over the last 15 years as a viable route to clinically-relevant natural products. This talk will feature recent successful examples of polyketide and isoprenoid natural products produced heterologously through E. coli. Emphasis will be placed on the technical challenges and strategies associated with functional gene transfer and expression within this alternative host.

4:50 Reception in the Exhibit Hall with Poster Viewing

6:00 End of Day



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