Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris

Application to intracellular production of Hepatitis B surface antigen

authored by

Chandrasekhar Gurramkonda, Ahmad Adnan, Thomas Gäbel, Heinrich Lünsdorf, Anton Ross, Satish Kumar Nemani, Sathyamangalam Swaminathan, Navin Khanna, Ursula Rinas

Abstract

Background: Hepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase 1 locus. Results: Large-scale production was optimized by developing a simple fed-batch process leading to enhanced product titers. Cells were first grown rapidly to high-cell density in a batch process using a simple defined medium with low salt and high glycerol concentrations. Induction of recombinant product synthesis was carried out using rather drastic conditions, namely through the addition of methanol to a final concentration of 6 g L^-1. This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the on-line signal of a flame ionization detector employed as methanol analyzer in the off-gas stream. Using this robust feeding protocol, maximum concentrations of ∼7 grams HBsAg per liter culture broth were obtained. The amount of soluble HBsAg, competent for assembly into characteristic virus-like particles (VLPs), an attribute critical to its immunogenicity and efficacy as a hepatitis B vaccine, reached 2.3 grams per liter of culture broth. Conclusion: In comparison to the highest yields reported so far, our simple cultivation process resulted in an ∼7 fold enhancement in total HBsAg production with more than 30% of soluble protein competent for assembly into VLPs. This work opens up the possibility of significantly reducing the cost of vaccine production with implications for expanding hepatitis B vaccination in resource-poor countries.

External Organisation(s)

International Centre for Genetic Engineering and Biotechnology
Helmholtz Centre for Infection Research (HZI)
Government College University Lahore
Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM)

Type

Article

Journal

Microbial cell factories

Volume

8

ISSN

1475-2859

Publication date

10.02.2009

Publication status

Published

Peer reviewed

Yes

ASJC Scopus subject areas

Biotechnology, Bioengineering, Applied Microbiology and Biotechnology

Sustainable Development Goals

SDG 3 - Good Health and Well-being

Electronic version(s)

https://doi.org/10.1186/1475-2859-8-13 (Access: Open)