Implementing Chemostat Fermentation of Pichia Pastoris Producing Recombinant HBsAg to Optimize Cell Density Affected by Methanol Rate

Abstract

High cell density fed-batch fermentation is the main strategy for recombinant hepatitis B surface antigen (rHBsAg) production. In this study, we employed short-term continuous fermentation to optimize the cell density of recombinant Pichia pastoris (P. pastoris). After reaching the maximum specified broth volume of 5 L in the fed-batch fermentation process, the operation mode was altered into the continuous mode with a dilution rate of 0.009 1/h. We used various values of methanol inflow to examine its impact as a limiting nutrient on cell density. After reaching the steady-state point, the continuous fermentation was stopped. The process's performance was evaluated based on titer, yield, productivity, and ease of process control. According to the results, the optimal methanol inflow in the pilot-scale fermentation process was 39.9 ml/h as the cell density increased from 363 g/l wet cell weight (WCW) in the fed-batch stage to 450 g/l WCW. We could successfully scale up the fermentation process with the biomass concentration of 450 g/l without having any major issues such as excessive heat dissipation or insufficient oxygen supply. This approach is a simple method for enhancing rHBsAg production efficiency in P. pastoris without requiring any new and complex facility.