1. Expression of EGFP Reporter Protein with a Recombinant Vaccinia Virus - Comparison of Microcarrier and Cell Suspension Based Bioreactor Systems 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 Nicole A. Bleckwenn 1,2,3, William Bentley 2,3, and Joseph Shiloach 1 ABSTRACT 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. 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 BACKGROUND CONCLUSIONS COMPARISON OF MICROCARRIER AND SUSPENSION SYSTEMS Growth Comparison in Bioreactors 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 Special Thanks to Bernard Moss and Pat Earl (NIAID, NIH) for providing VOTE components and Refine Technology, Co. for providing ATF™ System PARAMETER OPTIMIZATION Production with the Microcarrier System DO Levels in Spinner Flasks (by changes in surface area to volume ratio of media in flasks) Temperature in Spinner Flasks Microcarrier Culture Suspension Culture * 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. DO Levels in Bioreactor Effect of MOI on Expression in Spinner Flasks Protein Production Comparison in Bioreactors Vacuum Level Control Feed Pump Water Jacket Inlet Outlet D.O. pH Agit. Temp. Filtrate Pump Air Inlet Mesh Screen Module Diaphragm ATF Controller Bioreactor Setup for Microcarrier Culture Gas inlet into reactor headspace Level Control Feed Pump Water Jacket Inlet Outlet D.O. pH Agit. Temp. Hollow Fiber Unit Circulation Pump Permeate Pump Bioreactor Setup for Suspension Culture Gas inlet into headspace or liquid Specific Production Overall Production