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HBsAg Production in Methanol Controlled P. pastoris GS115 MutS Bioreactor Process

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Abstract:

When producing recombinant proteins with Pichia pastoris, cultivation parameters, such as induction temperature, dissolved oxygen level and residual methanol concentration play a crucial role in product biosynthesis and subsequent purification, therefore to maximize protein yields, the optimization of these parameters is imperative. Two different Pichia pastoris cultivation strategies for HBsAg VLP production in a 5 L stirred-tank bioreactor and the influence of different cultivation parameters on product yield were investigated. Residual methanol concentrations were controlled at low (>0.01 g/L), medium (1.5-2.0 g/L) and high (5.0-6.0 g/L) levels using a PI-based feed rate control algorithm based on the online methanol sensor signal. Product was purified using a novel and rapid purification method including steps of ammonium sulfate precipitation, size-exclusion chromatography and hydrophobic interaction chromatography. Employing an in-situ methanol sensor probe, the PI-based methanol feed rate control algorithm provided residual methanol concentration control with an average deviation of ±0.4 g/L from set-point value. Employing a cultivation protocol with an increased methanol concentration controlled at 6.0 g/L and a reduced DO level below 10 %, resulting in a final dry cell biomass concentration of 140 g/L and purified HBsAg VLPs yield of 186 mg/L. Developed purification method proved advantageous to other described methods, as it did not include time consuming extraction and centrifugation steps.

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Periodical:

Key Engineering Materials (Volume 903)

Pages:

40-45

DOI:

https://doi.org/10.4028/www.scientific.net/KEM.903.40

Citation:

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Online since:

November 2021

Authors:

Oskars Grigs*, Emīls Bolmanis, Andris Kazaks

Keywords:

Hepatitis B Surface Antigen, Methanol PI-Control, Pichia pastoris, Yeast Cultivation

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* - Corresponding Author

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