Continuous fermentation of recombinant Pichia pastoris Mut+ producing HBsAg: Optimizing dilution rate and determining strain-specific parameters

Abstract

There is a growing interest in the biopharmaceutical industry to enhance production efficiency via shifting batch to continuous manufacturing. In the previous study, we demonstrated that under identical operation condition, continuous fermentation of recombinant Pichia pastoris (P. pastoris) Mut+ producing intracellular hepatitis B surface antigen (HBsAg) has considerably higher efficiency compared to the conventional fed-batch process. In the current work, by examining various dilution rates, we further optimized the process efficiency, and also determined some strain-specific parameters. According to the results, continuous operation at the dilution rate of 0.015 1/h demonstrated the highest performance compared to other dilution rates of 0.009, 0.02, 0.25, 0.03 and 0.0417 1/h. In the optimum dilution rate, the average HBsAg titer, yield, specific and volumetric productivity were 283.9mgHBsAg/L, 0.71mgHBsAg/g MeOH, 0.0097mgHBsAg/g wet cell weight (WCW)/h and 4.26mgHBsAg/L/h, respectively. Compared to the continuous fermentation in the previous study, the volumetric and specific productivities were improved by the factors of 2.0 and 2.5, respectively. Following Herbert-Pirt linear relation between specific growth rate and methanol consumption rate, the obtained biomass yield and maintenance coefficient in the continuous fermentation were 1.22 (g WCW/g MeOH) and 0.008 (g MeOH/g WCW/h), respectively. The consistency in the process condition and efficiency without any genetic instability and contamination in the one-month operation- in each fermentation run- suggests that the studied recombinant strain could be one of the potential candidates for continuous biomanufacturing of hepatitis B vaccine.