Production of recombinant proteins by vaccinia virus in a microcarrier based mammalian cell perfusion bioreactor

Authors

  • Nicole A. Bleckwenn,

    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
    2. Department of Chemical Engineering, University of Maryland, College Park, Maryland
    3. Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, Maryland
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  • Hana Golding,

    1. Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland
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  • William E. Bentley,

    1. Department of Chemical Engineering, University of Maryland, College Park, Maryland
    2. Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, Maryland
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  • Joseph Shiloach

    Corresponding author
    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
    • 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.
    Search for more papers by this author

  • This article is a US Government work and, as such, is in the public domain in the United States of America.

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 × 106 to 4.4 × 106 cell/mL. A seed train expansion method using cells harvested from microcarrier culture and reseeding onto fresh carriers was developed. 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 μg/106 infected cells, was obtained at 50% DO and 31°C. 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 ID50 of 3.1 μg/mL, which is comparable to previous reports. Published 2005 Wiley-Periodicals, Inc.

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