The optimisation of recombinant protein production by animal cell cultures is important for the economic feasibility of these processes. Simultaneously with product yield, product authenticity is a crucial aspect to consider as it may per se affect the therapeutic value of such proteins. More defined culture media are being developed, particularly to ensure batch product consistency. A Chinese Hamster Ovary cell line (CHO 320) producing human interferon-γ (IFN-γ), a glycosylated protein, was chosen to investigate the effects of the culture environment on (I) cell growth, (2) product yield and (3) product authenticity. A statistical approach was used to identify important culture components for cell growth and IFN-γ production. When the concentration of the resulting positive variables was initially increased in culture, improvements of approximately 40% in both of these parameters were achieved; the glycosylation of IFN-γ was not affected. The former analysis also indicated that different stimuli were required for growth and production. Fed-batch feeding of glucose and glutamine, components depleted early from culture, did not prolong cell growth or IFN-γ production but the initial glycosylation pattern of IFN-γ was a function of glutamine concentration. Bovine serum albumin (BSA) was shown to have important role(s) in culture and cell growth was not possible in its absence. Pluronic F68, alone or in combination with a lipid mixture or linoleic acid, was able to restore cell growth in low BSA (1 mg/ml) cultures. However, IFN-γ production was significantly reduced and the extent of IFN-γ glycosylation also changed. These effects were related to: (1) BSA concentration, (2) BSA type, and ultimately, (3) lipid composition of the culture. The results reported in this thesis exhibit the necessity to consider the effects of the culture environment not only on cell growth and product yield but also on product authenticity throughout any optimisation process.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:309928 |
| Date | January 1993 |
| Creators | Lima e Castro, Paula Maria |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1317969/ |
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