Bethesda,MD,30.3.2004.ppt

Background and Rationale Recombinant proteins are essential research tools for ? assay development and screening ? structural biology ? antibody generation, selectivity assays...... In the post-genomic era translation of thousands of ORFs into proteins are a major challenge Technologies and processes increasing the throughput and the success rate in protein production are needed Novartis Research is currently building a Protein Production Center in which streamlined processes will be applied in a factory-like fashion. In parallel, an Intensive Care Approach is applied to generate proteins by non-generic means. Double-Track Strategy Target output: 400 tool proteins/year Quantity 1 - 100 mg Purity 80 % (application-dependent) “Protein Factory”: 300 proteins/year 3 standardized expression systems Streamlined, generic, partly automated processes “Intensive Care”: 100 proteins/year Difficult-to-express proteins Recombinant cell lines, membrane proteins Monoclonal and recombinant antibodies A Double-Track Process to Increase Success Rates Small Scale Expression Evaluation Why HEK.EBNA Cells? The Principle Why HEK.EBNA Cells? Advantages HEK.EBNA Expression Vectors Small Scale Expression in HEK.EBNA Cells Summary of Small Scale Expression Large Scale Transient Transfection (1) Large Scale Transient Transfection (2) Large Scale Transient Transfection (3) Large Scale Transient Transfection (4) Polyethylenimine-mediated Transfection A Transient Transfection Run….. ….in Multiparallel Fashion Cell/Supernatant Harvest and Cell Lysis Protein Purification A Few Examples To summarize……. Acknowledgements: The BTP team * PPCW Workshop NIH 3/28/2004 * EXA Protein Science Forum Horsham 18./19.11.2002 Bethesda,MD,30.3.2004 Sabine Geisse, Nicola di Maiuta, Thomas Cremer, Mario Henke  Novartis Institutes for Biomedical Research, Basle, Switzerland Transient Transfection into Eukaryotic Cells: An Alternative to Bacterial and Insect Cell Systems for the Rapid Generatio