High level production and purification of human interferon α2b in high cell density culture of Pichia pastoris

Enzyme and Microbial Technology
Atef AyedHéla Kallel

Abstract

Human interferon α2b gene was cloned in the methylotrophic yeast Pichia pastoris under the control of the AOX1 methanol inducible promoter. To optimise the volumetric productivity, we performed different fed-batch studies in a 5-L bioreactor. We demonstrated that hIFNα2b was highly sensitive to proteases activity during high cell density culture. The target protein was totally degraded 20h after the start of methanol feeding. Replacement of culture medium with fresh medium after glycerol fed-batch culture mode as well as medium enrichment with casamino acids at 0.1% and EDTA at 10mM, had significantly improved hIFNα2b expression and prevented its proteolysis. Moreover, to further improve hIFNα2b production, three different methanol fed-batch strategies had been assayed in high cell density culture. The optimal strategy resulted in a production level of 600mg/l while residual methanol level was maintained below 2g/l. Clarification of culture supernatant through a 0.1μm hollow fiber cartridge showed that almost 95% of the target protein was retained within the retentate. Triton X-100 or NaCl addition to the culture harvest before microfiltration had improved the recovery yield of this step. rhIFNα2b was further purified by cation...Continue Reading

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Citations

Nov 5, 2013·Biotechnology Letters·Omid BayatRaha Abdul Rahim
Feb 18, 2010·Protein Expression and Purification·Shardul SalunkheSriram Padmanabhan
Jun 12, 2016·Microbial Cell Factories·Christopher P LandowskiMarkku Saloheimo
Mar 10, 2017·Bioscience, Biotechnology, and Biochemistry·Mohsin Ahmad KhanTayyab Husnain
Mar 30, 2017·Journal of Industrial Microbiology & Biotechnology·Xiao-Dong WangYan Xu
Oct 10, 2018·Applied Microbiology and Biotechnology· AdivitiyaYogender Pal Khasa

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