Pichia pastoris (P. pastoris) expression systems are gaining increased interest in industry due to their ability to achieve high cell densities and production levels. This thesis aims to characterise the following critical elements of P. pastoris upstream bioprocessing: cell growth, cell productivity and bioprocess monitoring. Methanol is the principle carbon source and gene expression induction agent in most P. pastoris fermentation strategies. Monitoring of methanol levels during fermentation enables cell growth and productivity to be optimised, whilst methanol toxicity is avoided. A novel approach to at-line methanol monitoring was investigated, seeking to exploit the chromogenic reactivity of dehydroascorbic acid (DHAA) with methanol. Recombinant variants of the human reporter enzyme, placental alkaline phosphatase (PLAP) were used as a model fermentation product, and the performance of two mechanistically distinct commercial assays was compared for their applicability to high cell density process streams. In total four new P. pastoris strains expressing PLAP variants were successfully created and the impact of methanol carbon source feed strategies on the strain performance investigated in terms of growth rate and recombinant protein production. A 2-fold increase in rate of methanol feed resulted in a final biomass increase of about 40% and in a volumetric productivity decrease of about 75%. A novel method, combining phases of low and high methanol feed rates, matched but did not exceed conventional methanol feed rate strategies.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:634623 |
| Date | January 2014 |
| Creators | Randone, P. |
| 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/1456760/ |
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