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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 …