Two-stage glycerol feeding for enhancement of recombinant hG-CSF production in a fed-batch culture of Pichia pastoris

Recombinant hG-CSF was expressed in Pichia pastoris under the control of the AOX1 promoter. In this study, the glycerol feeding rate was adjusted to achieve the maximum attainable specific growth rate before induction. Using a two-stage glycerol feeding method, the specific growth rate was changed from a maximum value of 0.21 h⁻¹ (at the beginning of feeding) to 0.15 h⁻¹ prior to induction. With this approach, the final dry cell wt and rhG-CSF yield achieved was close to 120 g l⁻¹ and 320 mg l⁻¹, respectively. Our study found that the two-stage feeding method allowed the overall productivity of rhG-CSF to increase 2.9 times that of the conventional fed-batch method.