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Protein Production Development with Recombinant Vaccinia Virus

dc.contributor.advisorBentley, William Een_US
dc.contributor.authorBleckwenn, Nicole Aleeceen_US
dc.date.accessioned2004-06-04T05:36:14Z
dc.date.available2004-06-04T05:36:14Z
dc.date.issued2004-04-29en_US
dc.identifier.urihttp://hdl.handle.net/1903/1416
dc.description.abstractThe 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.en_US
dc.format.extent1408248 bytes
dc.format.mimetypeapplication/pdf
dc.language.isoen_US
dc.titleProtein Production Development with Recombinant Vaccinia Virusen_US
dc.typeDissertationen_US
dc.relation.isAvailableAtDigital Repository at the University of Marylanden_US
dc.relation.isAvailableAtUniversity of Maryland (College Park, Md.)en_US
dc.contributor.departmentChemical Engineeringen_US
dc.subject.pqcontrolledEngineering, Chemicalen_US
dc.subject.pquncontrolledvacciniaen_US
dc.subject.pquncontrolledbioreactoren_US
dc.subject.pquncontrolledEGFPen_US
dc.subject.pquncontrolledgp120en_US
dc.subject.pquncontrolledproductionen_US
dc.subject.pquncontrolledperfusionen_US


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