Production of Recombinant Proteins by Vaccinia Virus in a Microcarrier Based Mammalian Cell Perfusion Bioreactor Nicole A. Bleckwenn, 1,3,4 Hana Golding, 2 William E. Bentley, 3,4 Joseph Shiloach 1 1 Chief, Biotechnology Unit, NIDDK, National Institutes of Health, DHHS, Bldg. 14A Rm. 173, MSC 5522, 9000 Rockville Pike, Bethesda, Maryland; telephone: 301-496-9719; fax: 301-451-5911; e-mail: yossi@nih.gov 2 Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 3 Department of Chemical Engineering, University of Maryland, College Park, Maryland 4 Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, Maryland Received 13 May 2004; accepted 3 December 2004 Published online 27 April 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20423 Abstract: The HeLa cell-vaccinia virus expression system was evaluated for the production of recombinant proteins (enhanced green fluorescent protein (EGFP) and HIV envelope coat protein, gp120) using microcarriers in 1.5 L perfused bioreactor cultures. Perfusion was achieved by use of an alternating tangential flow device (ATF), increasing the length of the exponential phase by 50 h compared to batch culture and increasing the maximum cell density from 1.5  10 6 to 4.4  10 6 cell/mL. A seed train expansion method using cells harvested from microcar- rier culture and reseeding onto fresh carriers was devel- oped. EGFP was first used as a model protein to study process parameters affecting protein yield, specifically dissolved oxygen (DO) and temperature during the production phase. The highest level of EGFP, 12  1.5 mg/ 10 6 infected cells, was obtained at 50% DO and 318C. These setpoints were then used to produce glycoprotein, gp120, which was purified and deglycosylated, revealing a significant amount of N-linked glycosylation. Also, biological activity was assayed, resulting in an ID 50 of 3.1 mg/mL, which is comparable to previous reports. Published 2005 Wiley Periodicals, Inc. { Keywords: vaccinia; bioreactor; microcarrier; ATF; EGFP; gp120 INTRODUCTION Recombinant protein expression from cell culture can be accomplished by stable or transient integration of DNA into the host cells. Viral vectors provide efficient transient expression and have been used widely (e.g., insect cells, insect larvae). The baculovirus has proven to be very effective at producing large quantities of foreign proteins (Bentley et al., 1994; Cha et al., 1999; Chung et al., 1993; Licari and Bailey, 1991; Lindsay and Betenbaugh, 1992; Wickham and Nemerow, 1993). Generally, complex proteins made with this system are not processed in the same manner as in mammalian cells, without significantly altering the host cells’ post-translational processing machinery (Ailor and Betenbaugh, 1999; Ailor et al., 1999, 2000; Fang et al., 2000; Percival et al., 1997). When proper post-translational modifications are necessary, for example, for proteins used as therapeutic agents (Eckhardt et al., 2002; Fenouillet and Jones, 1996; Li et al., 1993), it may be advantageous to use a mammalian derived host cell line. Our lab has postulated that a viral system used with mammalian cell culture, a more complex system with innate capabilities for complex post-translational processing, might lead to increased levels of protein production compared to stable integration of foreign DNA into the host cell. Vaccinia virus, an orthopoxvirus related to the virus that causes smallpox, was chosen as the viral vector due to its unique characteristics. This virus has a wide host range (Broder and Earl, 1999) and its own transcriptional machinery is brought into the cytoplasm of the infected cell (Moss, 1996). This feature allows the recombinant protein to be transcribed in the cytoplasm, eliminating nuclear transport requirements for the recombinant DNA and mRNA transcripts. Bypassing these potentially rate-limiting transport steps during cell line development and protein production may increase the ability of this system to produce larger proteins than those systems without this feature. For example, it is speculated that the capacity for gene insertions in vaccinia virus is up to 25,000 bp of DNA, exceeding the capacity of other mammalian expression vectors (Moss, 1991; Smith and Moss, 1983). Also, when comparing short term methods of Published 2005 Wiley Periodicals, Inc. { This article is a US Government work and, as such, is in the public domain in the United States of America. Correspondence to: Joseph Shiloach Contract grant sponsor: NIDDK, National Institutes of Health, DHHS, Bethesda, MD