Prevention of human granulocyte colony-stimulating factor protein aggregation in recombinant Pichia pastoris fed-batch fermentation using additives

rhG-CSF (recombinant human granulocyte colony-stimulating factor) was expressed in the yeast Pichia pastoris under the control of the AOX1 (alcohol oxidase 1) promoter. The production of rhG-CSF was induced by switching from growth on glycerol to growth on methanol. In the induction phase, the methanol feed rate had a significant effect on the specific expression rate of rhG-CSF. A constant feed rate of 16 ml . h(-1) . l(-1) was found to be optimal for a high specific expression rate of rhG-CSF (0.058 mg(-1) . h(-1) . g DCW-1; DCW is dry cell weight). Under this condition, a maximum concentration of 300 mg/l of rhG-CSF and the expression yield of 0.6 mg of rhG-CSF/g of methanol were attained. However, the secreted rhG-CSF was shown to exist as aggregates in the culture broth, owing to hydrophobic interactions. To prevent undesirable protein aggregation, the presence of additional additives in the R pastoris culture medium was investigated. Among seven additives tested, Tween 20, Tween 80 and betaine exhibited the best results in respect of preventing the formation of rhG-CSF protein aggregates.