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

Recombinant protein production in Pichia pastoris is based on alcohol oxidase promoterswhich 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) arefocusing on methanol mixed feeding strategies. This work aimed to develop a new experimentalmethod and compare the effect of various fractions of sorbitol in mixed feeding strategy withstepwise addition of methanol to maximize the productivity of human growth hormone.Accordingly, fed-batch culture performed with a mixed feed of sorbitol/methanol. Sorbitol atconcentrations of 30, 40, 50 and 60 gram per liter was added in batch-wise mode to the mediumat the beginning of induction phase and the rate of methanol addition was increased stepwiseduring the first 12 h of production and then kept constant. In order to understand the effects ofvarious co-substrate feeding strategies on P. pastoris and its production of hGH, cell mass, drycell weight, total protein, hGH expression level and hGH concentration were analyzed and theresults 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 significantlyincrease 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/Lfollowing 30 h induction.