Wang, Baolong; (2019) Development of New Cultivation Strategies to Enhance Heterologous Protein Production in Pichia Pastoris. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Growth of the biopharmaceutical market and advances in host strain engineering have fuelled the application of Pichia pastoris in recombinant protein production. Production of recombinant protein in P. pastoris is commonly induced by continuous methanol feeding. However, methanol induction challenges scale-up of cultivation due to high oxygen requirement and substantial heat generation. Developing novel induction strategies to minimize methanol consumption is desirable. This thesis compared the standard methanol induction with a sorbitol/methanol mixed induction strategy and developed the induction method at large scale by using oxygen transfer rates (OTR) as a scale-down criterion. Influences of sorbitol/methanol mixed induction on product recovery were also studied using scale-down methodology. Compared to standard methanol induction, substituting 50% (C-mol/C-mol) methanol with sorbitol improved cell viability from 92.8±0.3% to 97.7±0.1% but reduced product yield from 1.65±0.03 g•L-1 to 1.12±0.07 g•L-1. Oxygen uptake rate was reduced from 241.4±15.0 mmol•L-1•h-1 to 145.5±4.8 mmol•L-1•h-1 by using the mixed induction. Proteomics study showed that supernatant from mixed induction contained fewer host cell proteins (72 versus 96) and fewer types of protease (1 versus 3). OTR expected in the large scale bioreactors was used as scale-down criterion at one litre. By measuring oxygen transfer coefficient (kLa) of the small bioreactor, a fermentation process with OTR of 150 mmol•L-1•h-1 was defined. Standard methanol induction would cause oxygen depletion and methanol accumulation in the medium. Residual methanol concentration apparently influenced the cell growth and product expression. The biomass and product yield reached 108.3 g•L-1 and 1.1 g•L-1 when the residual methanol concentration was below 5 g•L-1, whereas they were reduced to 75.5 g•L-1 and 0.87 g•L-1 at a higher concentration (5~10 g•L-1). Decreasing methanol feeding rate avoided the oxygen depletion. However, the biomass and product yield were reduced to 92.2 g•L-1 and 0.85 g•L-1. Partially replacing methanol with sorbitol enhanced the biomass to 130 g•L-1 but the product yield was not enhanced. An ultra scale-down approach enabled the prediction of cell culture dewatering in pilot and industrial scale centrifuges. It was found that the cell culture from mixed induction had higher centrifugal dewatering levels (84.5±3.3% versus 78.1±3.9%), which was likely to be attributed to the decrease of cell diameter during induction.

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