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    Protein Production Development with Recombinant Vaccinia Virus

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    No. of downloads: 1859

    Date
    2004-04-29
    Author
    Bleckwenn, Nicole Aleece
    Advisor
    Bentley, William E
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    Abstract
    The vaccinia virus expression system was developed into a scaleable recombinant protein production process in perfused mammalian cell culture. Growth of anchorage dependent HeLa cells on microcarriers and the suspension adapted HeLa S3 cell line were studied in bioreactor cultures utilizing the ATF System or hollow fiber filter, respectively, for perfusion. Recombinant vaccinia virus expressing enhanced green fluorescent protein (EGFP) as a model protein was used to study the effects of several process parameters on expression. These included multiplicity of infection (MOI), volume during infection, serum concentration during infection, inducer concentration, timing of inducer addition relative to infection, and dissolved oxygen and temperature during the protein production phase. Increases in protein yield were made as each of these parameters was studied. The microcarrier based system reached 20 mg/l EGFP while the suspension based system achieved 27 mg/l under the conditions found through experiment. A second virus containing the gene for gp120, an HIV envelope coat protein with complex post-translational modifications, was produced in microcarrier based bioreactor culture with HeLa cells. The protein produced was purified and analyzed for post-translational modifications which found that half of the molecular weight was contributed through N-linked glycans. The reactor culture produced 10.5 mg/l gp120 at 96 hours post infection with an ID(50) of 3.1 µg/ml. A survey of expression, using both EGFP and gp120 expressing viruses, was conducted on several mammalian cell lines which may be more appropriate for commercial manufacturing processes. Results varied, depending on the protein produced, with HeLa cells producing the most EGFP and BS-C-1 the most gp120. 293 cells performed fairly well in both cases and their use in other manufacturing processes and ability to grow in serum-free suspension culture lead to a recommendation that they be considered for further process development. These studies have provided insight into the vaccinia virus expression system as a potentially large-scale production method for complex human proteins. Further optimization of the process could continue to increase the yields and potentially bring this viral process into the arena of available technologies for production.
    URI
    http://hdl.handle.net/1903/1416
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    • Chemical and Biomolecular Engineering Theses and Dissertations
    • UMD Theses and Dissertations

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    DRUM is brought to you by the University of Maryland Libraries
    University of Maryland, College Park, MD 20742-7011 (301)314-1328.
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