Transient protein production by cultured mammalian cells from transfected episomal DNA is frequently used in bioindustry to generate small quantities of candidate therapeutic products during early stages of process development. However, transient production processes typically exhibit low productivity, limiting their use at scale. In this thesis, three distinct but complementary approaches were evaluated for the de novo design of a high productivity scaleable transient production process starting with the discrete raw materials: transfection reagent, Chinese hamster ovary cell line, plasmid DNA and chemically defined medium. (1) Optimisation of CHO host cell transfection: The optimal combination of continuous basal parameters underpinning polyethylenimine (PEI) mediated transfection (relative concentrations of PEI, plasmid DNA and cells) was determined utilising Design of Experiments (DoE) methodology. Optimum transfection conditions were cell line specific - highly dependent upon resistance to PEI cytotoxicity. Comparing different CHO cell hosts operating at their unique optima, variations in specific productivity were limited by the rate of polyplex endocytosis. (2) Modulation of the cell culture environment: Combinations of environmental variables were evaluated using factorial screening to determine an optimal cell culture regime for transient production. For the CHO cells used in this study, the addition of valproic acid, recombinant insulin-like growth factor and a reduced culture temperature were found to interact synergistically to maximise recombinant product yield at an increased cell concentration. (3) Production process design: Utilising response surface modelling to determine key process interactions, transient transfection and medium environment optima were effectively combined to create an intensified, high cell density process exhibiting a five-fold increase in volumetric titre. Combining these approaches, volumetric yield for a transient monoclonal antibody production process was increased from 2 mg L-1 to > 90 mg L-1 - the highest transient volumetric titre achieved with un-genetically modified CHO cells in a chemically defined environment to date.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:578708 |
| Date | January 2011 |
| Creators | Thompson, Ben C. |
| Publisher | University of Sheffield |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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