Quantitative comparison of dynamic physiological feeding profiles for recombinant protein production with Pichia pastoris

Pichia pastoris is widely used for the production of recombinant proteins in industrial biotechnology. In general, industrial production processes describe fed-batch processes based on the specific growth rate. Recently, we introduced the specific substrate uptake rate (q (s)) as a novel parameter to design fed-batch strategies for P. pastoris. We showed that a dynamic feeding strategy where the feed was adjusted in steps to the maximum specific substrate uptake rate was superior to more traditional strategies in terms of specific productivity. In the present study, we compare three different dynamic feeding strategies based on q (s) for a recombinant P. pastoris strain with respect to cell physiology, methanol accumulation, productivity and product quality. By comparing (A) a feeding profile at constant high q (s), (B) a periodically adjusted feeding profile for a stepwise q (s) ramp, and (C) a feeding profile at linear increasing q (s), we evaluated potential effects of the mode of feeding. Although a dynamic feeding strategy with stepwise increases of q (s) to q (s max) resulted in the highest specific productivity, a feeding profile where the feeding rate was stepwise increased to a constant high q (s) value was superior in terms of the amount of active enzyme produced and in the amount of accumulated methanol. Furthermore, this feeding strategy could be run automatically by integrating an online calculator tool, thus rendering manual interventions by the operator unnecessary.