Scaleable Protein Production in Anchorage Dependent Mammalian Cells

1. Induced Infected Uninfected lac O EMC SLO Target T7 gene I PL PE/L lac I PT7 PE/L gpt TT Fluorescence Inactive Repressor Target Gene Expression Light lac O EMC SLO Target T7 gene I PL PE/L lac I PT7 PE/L gpt TT Active Repressor Uninfected Scaleable Protein Production in Anchorage Dependent Mammalian Cells Nicole A. Bleckwenn123, William Bentley23, and Joseph Shiloach1 ABSTRACT RESULTS – Protein Expression 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 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. 6-Well Plate Infection Experiments RESULTS – Cell Growth Parameters Tested Result Multiplicity of infection (MOI, pfu/cell) 1.0 Volume during infection Lowest Possible (0.5 mL) Serum concentration during infection None Infection duration 1 hr Inducer concentration 1.0 mM IPTG Inducer addition timing At time of infection Bioreactor Setup for Microcarrier Culture Vacuum Diaphragm Mesh Screen Module Air Inlet ATF Controller BACKGROUND MOI Evaluation in Spinner Flasks Filtrate Pump Project Scope Picture of Bioreactor D.O. Temp. • 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 Level Control pH Agit. Feed Pump Infection in 1.5 L Bioreactor Water Jacket VOTE* Expression System Inlet Outlet Uninduced 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 FUTURE WORK 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 Recombinant Vaccinia Virus Construction EGFP-His6 EGFP vT7lacOI Infect pVOTE.2 -EGFP-His6 pEGFP-N1 EGFP-His6 Plaque Purify Transfect pSecTag2A -EGFP Insert EGFP Insert EGFP-His6 Infected only vEGFP-His6 HeLa Cells Special Thanks to Bernard Moss and Pat Earl (NIAID, NIH) for providing VOTE components Refine Technologies for providing ATF System Infected and Induced * 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.