Recombinant hepatitis B core antigen (HBcAg) molecules, produced in heterologous expression systems, self-assemble into highly homogenous and non-infectious virus-like particles (VLPs) that are under extensive research for biomedical applications. HBcAg production in the methylotrophic yeast P. pastoris has been well documented; however, productivity screening under various residual methanol levels has not been reported for bioreactor processes. HBcAg production under various excess methanol levels of 0.1, 1.0 and 2.0 g L-1 was investigated in this research. Results indicate that, under these particular conditions, the total process and specific protein yields of 876-1308 mg L-1 and 7.9-11.2 mg g(DCW)(-1), respectively, were achieved after 67-75 h of cultivation. Produced HBcAg molecules were efficiently purified and the presence of highly immunogenic, correctly formed and homogenous HBcAg-VLPs with an estimated purity of 90% was confirmed by electron microscopy. The highest reported HBcAg yield of 1308 mg L-1 and 11.2 mg g(DCW)(-1) was achieved under limiting residual methanol concentration, which is about 2.5 times higher than the next highest reported result. A PI-algorithm-based residual methanol concentration feed rate controller was employed to maintain a set residual methanol concentration. Finally, mathematical process models to characterise the vegetative, dead and total cell biomass (X-v, X-d and X), substrate (Glycerol and Methanol) concentration, reactor volume (V), and product (HBcAg) dynamics during cultivation, were identified. A rare attempt to model the residual methanol concentration during induction is also presented.
