1. Scaleable Protein Production in Anchorage Dependent Mammalian Cells 1 Biotechnology Unit, NIDDK,National Institutes of Health, Bethesda, MD 2 Center for Biosystems Research, UMBI, College Park, MD 3 Department of Chemical Engineering, UMCP, College Park, MD Nicole A. Bleckwenn 123, William Bentley 23, and Joseph Shiloach 1 ABSTRACT Recombinant protein production is a common method for providing clinical and commercial quantities of human therapeutic agents. Methods of production include recombinant bacterial fermentations, transfected or transformed cell culture, and virally infected cell culture. A potential protein production method with anchorage dependent mammalian cells has been evaluated. The system utilizes a recombinant vaccinia virus with the VOTE expression system that can potentially be used to produce large, highly glycosylated recombinant proteins, which may be difficult to produce by other means. A reporter protein, enhanced green fluorescent protein (EGFP), has been used to study the characteristics of this protein production method. Growth, infection and production parameters such as multiplicity of infection, culture volume during infection, infection duration, inducer concentration and timing of inducer addition were studied in monolayer culture. These results were then used to establish conditions for studies in microcarrier spinner flask and 1.5 L bioreactor cultures. Production processes utilizing the selected conditions will be described, together with an evaluation of the expression system. Objective - Develop an alternative protein production process with recombinant vaccinia virus Necessary components – Vaccinia Virus Transcription occurs in cytoplasm of infected cell Wide host range VOTE expression system for high, T7 promoter controlled expression – High cell density HeLa cells – human cervical adenocarcinoma Attachment dependent – growth on microcarriers Develop bioreactor strategy for growth Study infection process and parameters affecting expression Project Scope BACKGROUND VOTE * Expression System * Ward, G. A., Stover, C. K., Moss, B., and Fuerst, T. R. (1995). Stringent Chemical and Thermal Regulation of Recombinant Gene- Expression By Vaccinia Virus Vectors in Mammalian-Cells. Proceedings of the National Academy of Sciences of the United States of America 92, 6773-6777. Recombinant Vaccinia Virus Construction vT7lacOI HeLa Cells Transfect Infect vEGFP-His 6 Plaque Purify Insert EGFP-His 6 pVOTE.2 -EGFP-His 6 EGFP-His 6 pEGFP-N1 EGFP pSecTag2A -EGFP EGFP-His 6 Insert EGFP Infected and Induced Infected only Uninfected Vacuum Level Control Feed Pump Water Jacket Inlet Outlet D.O. pH Agit. Temp. Filtrate Pump Air Inlet Mesh Screen Module Diaphragm ATF Controller Parameters TestedResult Multiplicity of infection (MOI, pfu/cell)1.0 Volume during infectionLowest Possible (0.5 mL) Serum concentration during infectionNone Infection duration1 hr Inducer concentration1.0 mM IPTG Inducer addition timingAt time of infection InfectedUninfected Fluorescence Light FUTURE WORK MOI Evaluation in Spinner Flasks RESULTS – Cell Growth Bioreactor Setup for Microcarrier Culture ATF Unit Mechanism of Action A view through the sight glass as the diaphragm moves through an exhaust and pressure cycle Batch versus ATF Feeding Strategy 6-Well Plate Infection Experiments RESULTS – Protein Expression Infection in 1.5 L Bioreactor lac OEMCSLO Induced Inactive Repressor Target Gene Expression T7 gene IgptPLPL lac IP E/L TT P T7 P E/L Target Active Repressor T7 gene I lac O gptPLPL lac IP E/L TT EMCSLO P T7 P E/L Target Uninduced Cytodex 3 Microcarriers With Attached HeLa Cells More reactor studies – Dissolved oxygen, temperature, feeding strategy Test production in other cell lines – MRC-5, Vero, HEK 293, etc. Try other proteins (gp120, hGC-1) Verify post-translational processing Special Thanks to Bernard Moss and Pat Earl (NIAID, NIH) for providing VOTE components Refine Technologies for providing ATF System Picture of Bioreactor