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Bioreactor Setup for Microcarrier Culture. Vacuum. Diaphragm. Mesh Screen Module. Air Inlet. ATF Controller. Filtrate Pump. D.O. Temp. Level Control. pH. Agit. Feed Pump. Gas inlet into reactor headspace. Water Jacket. Inlet Outlet. Bioreactor Setup for Suspension Culture.
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Bioreactor Setup for Microcarrier Culture Vacuum Diaphragm Mesh Screen Module Air Inlet ATF Controller Filtrate Pump D.O. Temp. Level Control pH Agit. Feed Pump Gas inlet into reactor headspace Water Jacket Inlet Outlet Bioreactor Setup for Suspension Culture Temp. Gas inlet into headspace or liquid D.O. Level Control pH Agit. Feed Pump Hollow Fiber Unit Permeate Pump Water Jacket Inlet Outlet Circulation Pump Expression of EGFP Reporter Protein with a Recombinant Vaccinia Virus - Comparison of Microcarrier and Cell Suspension Based Bioreactor Systems Nicole A. Bleckwenn1,2,3, William Bentley2,3, and Joseph Shiloach1 ABSTRACT 1 Biotechnology Unit, NIDDK, National Institutes of Health, DHHS, Bethesda, MD 2 Center for Biosystems Research, UMBI, College Park, MD 3 Department of Chemical Engineering, UMCP, College Park, MD A recombinant vaccinia virus expression system was studied as an alternative method to produce recombinant proteins in a scaleable bioreactor system. A recombinant vaccinia virus containing the gene for the reporter protein, enhanced green fluorescent protein (EGFP), was used to study the system parameters that affect final protein expression. Evaluations of a microcarrier based method for adherent cell growth and infection in a bioreactor were performed. Controllable parameters such as temperature and dissolved oxygen level during production were tested to observe the effect on the level of protein production. Additionally, a cell suspension system was compared to the microcarrier method to determine if the levels of cell growth, viral infection, and ultimately protein expression could be maintained with a simpler production scheme. COMPARISON OF MICROCARRIER AND SUSPENSION SYSTEMS Effect of MOI on Expression in Spinner Flasks Microcarrier Culture Suspension Culture BACKGROUND • Objective - Develop a protein production process with recombinant vaccinia virus • Vaccinia Virus • Transcription occurs in cytoplasm of infected cell • Wide host range • VOTE* inducible expression system for high, T7 promoter controlled expression • Utilize previously constructed recombinant virus containing the gene for the reporter protein, enhanced green fluorescent protein (EGFP), to examine expression characteristics • High Cell Density • HeLa and HeLa S3 cells – human cervical adenocarcinoma • Compare attachment dependent growth on microcarriers to suspension growth • Develop bioreactor strategies for growth • Study infection processes for these two types of systems Growth Comparison in Bioreactors PARAMETER OPTIMIZATION Production with the Microcarrier System DO Levels in Bioreactor DO Levels in Spinner Flasks (by changes in surface area to volume ratio of media in flasks) Protein Production Comparison in Bioreactors Specific Production Overall Production Temperature in Spinner Flasks CONCLUSIONS • Higher dissolved oxygen and lower temperature during production increase expression levels • Both microcarrier and suspension systems can produce proteins • MOI above 1 appears to be more detrimental to suspension production than microcarrier production • Microcarrier system has a higher specific production • Suspension system has a higher overall production due to a higher cell density * 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. Special Thanks to Bernard Moss and Pat Earl (NIAID, NIH) for providing VOTE components and Refine Technology, Co. for providing ATF™ System