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Evaluation of Production Parameters with the Vaccinia Virus Expression System Using Microcarrier Attached HeLa Cells

Authors

  • Nicole A. Bleckwenn,

    1. Biotechnology Unit, NIDDK, National Institutes of Health, DHHS, Bldg 14A Rm 176, 9000 Rockville Pike, Bethesda, Maryland 20892
    2. Dept. of Chemical Engineering, University of Maryland, College Park, MD.
    3. Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, MD.
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  • William E. Bentley,

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

    Corresponding author
    1. Biotechnology Unit, NIDDK, National Institutes of Health, DHHS, Bldg 14A Rm 176, 9000 Rockville Pike, Bethesda, Maryland 20892
    • Biotechnology Unit, NIDDK, National Institutes of Health, DHHS, Bldg 14A Rm 176, 9000 Rockville Pike, Bethesda, Maryland 20892
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Abstract

Parameters that affect production of the recombinant reporter protein, EGFP, in the T7 promoter based VOTE vaccinia virus-HeLa cell expression system were examined. Length of infection phase, inducer concentration, and timing of its addition relative to infection were evaluated in 6-well plate monolayer cultures. One hour infection with 1.0 mM IPTG added at the time of infection provided a robust process. For larger scale experiments, anchorage-dependent HeLa cells were grown on 5 g/L Cytodex 3 microcarriers. The change to this dynamic culture environment, with cell-covered microcarriers suspended in culture medium in spinner flasks, suggested a re-examination of the multiplicity of infection (MOI) for this culture type that indicated a need for an increase in the number of virus particles per cell to 5.0, higher than that needed for complete infection in monolayer tissue flask culture. Additionally, dissolved oxygen level and temperature during the protein production phase were evaluated for their effect on EGFP expression in microcarrier spinner flask culture. Both increased dissolved oxygen, based on surface area to volume (SA/V) adjustments, and decreased temperature from 37 to 31 °C showed increases in EGFP production over the course of the production phase. The level of production achieved with this system reached approximately 17 μg EGFP/106 infected cells.

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