Design of Methanol Feed Control in Pichia pastoris Fermentations Based upon a Growth Model

The methylotrophic yeast Pichia pastoris is an effective system for recombinant protein productions that utilizes methanoi as an inducer, and also as carbon and energy source for a Mut (methanoi utilization plus) strain. Pichia fermentation is conducted in a fed-batch mode to obtain a high cell density for a high productivity. An accurate methanoi control is required in the methanoi fed-batch phase (induction phase) in the fermentation. A simple "on— off" control strategy is inadequate for precise control of methanoi concentrations in the fermentor. In this paper we employed a PID (proportional, integral and derivative) control system for the methanol concentration control and designed the PID controller settings on the basis of a Pichia growth model. The closed-loop system was built with four components: PID controller, methanoi feed pump, fermentation process, and methanoi sensor. First, modeling and transfer functions for all components were derived, followed by frequency response analysis, a powerful method for calculating the optimal PID parameters Kc (controller gain), TI (controller integral time constant), and TB (controller derivative time constant). Bode stability criteria were used to develop the stability diagram for evaluating the designed settings during the entire methanoi fed-batch phase. Fermentations were conducted using four Pichia strains, each expressing a different protein, to verify the control performance with optimal PID settings. The results showed that the methanoi concentration matched the set point very well with only small overshoot when the set point was switched, which indicated that a very good control performance was achieved. The method developed in this paper is robust and can serve as a framework for the design of other PID feedback control systems in biological processes.