Evaluation of Sorbitol-Methanol Co-Feeding Strategy on Production of Recombinant Human Growth Hormone in Pichia Pastoris

Document Type: Research article

Authors

1 Pharmaceutical sciences research center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 Food Safety Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

3 Department of Pharmaceutics, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

4 Department of Physiology and Pharmacology, Pasteur Institute of Iran, Tehran, Iran.

5 Department of Pharmaceutics, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran.

6 Pharmaceutical Sciences Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

7 Department of Pharmaceutics, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Abstract

Recombinant protein production in Pichia pastoris is based on alcohol oxidase promoters
which are regulated by methanol. However, the use of methanol has several disadvantages,
which is why current trends in bioprocess development with Pichia pastoris (P. pastoris) are
focusing on methanol mixed feeding strategies. This work aimed to develop a new experimental
method and compare the effect of various fractions of sorbitol in mixed feeding strategy with
stepwise addition of methanol to maximize the productivity of human growth hormone.
Accordingly, fed-batch culture performed with a mixed feed of sorbitol/methanol. Sorbitol at
concentrations of 30, 40, 50 and 60 gram per liter was added in batch-wise mode to the medium
at the beginning of induction phase and the rate of methanol addition was increased stepwise
during the first 12 h of production and then kept constant. In order to understand the effects of
various co-substrate feeding strategies on P. pastoris and its production of hGH, cell mass, dry
cell weight, total protein, hGH expression level and hGH concentration were analyzed and the
results compared with the basic feeding protocol using one-way analysis of variance (ANOVA).
According to the obtained results, sorbitol at a concentration of 50 g/L could significantly
increase the production yield. Under such optimal conditions cell biomass was 108 g/L (DCW),
total protein was 0.807 g/L, expression level was 83.1% and rhGH concentration was 0.667 g/L
following 30 h induction.

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