Enhancing HSA-GCSFm fusion protein production by Pichia pastoris with an on-line model-based exponential and DO-stat control modes

HSA-GCSF(m) fusion protein production by recombinant Pichia. pastoris with traditional DO-stat or methanol ON-OFF feeding strategy often suffers problems such as a limited methanol consumption, severe intracellular methanol accumulation, low protein titer etc. To solve these questions, an on-line model-based exponential and DO-stat control modes by controlling the oscillation bandwidths of dissolved oxygen concentration (DO) at a narrower and stable level (similar to 20%) was proposed. When using this strategy, 1) the accumulations of intracellular toxic methanol and formaldehyde were maintained at almost zero level by controlling DO at sufficient levels; 2) severe continuous oscillations in DO was effectively solved and thus methanol feeding/consumption rate could be increased from 0.0237-0.0281 g-MeOH/g-DCW/h to 0.0324 g-MeOH/g-DCW/h; 3) the lowest distribution of methanol to cells maintenance (0.0126 g-MeOH/g-DCW/h vs 0.0195 g-MeOH/g-DCW/h) was achieved and P. pastoris cells mortality decreased from 17.88% to 27.84-12.92% at the end of methanol induction phase; 4) the activities of the major enzymes involved in methanol metabolism, namely alcohol oxidase-AOX, formaldehyde dehydrogenase-FLD and formate dehydrogenase-FDH were largely activated; 5) maximum HSA-GCSF(m) concentration reached the highest level of 0.44 g/L, which was increased 46-238% compared to the control.