Optimization of cell density and dilution rate in Pichia pastoris continuous fermentations for production of recombinant proteins.

This paper provides an approach for optimizing the cell density (Xc) and dilution rate (D) in a chemostat for a Pichia pastoris continuous fermentation for the extracellular production of a recombinant protein, interferon tau (INF-tau). The objective was to maximize the volumetric productivity (Q, mg INF-tau l(-1) h(-1)), which was accomplished using response surface methodology (RSM) to model the response of Q as a function of Xc and D within the ranges 150< or = Xc < or =450 g cells (wet weight) l(-1) and 0.1 microm< or = D< or =0.9 microm (microm=0.0678 h(-1), the maximum specific growth rate obtained from a fed-batch phase controlled with a methanol sensor). The methanol and medium feed rates that resulted in the desired Xc and D were determined based on the mass balance. From the RSM model, the optimal Xc and D were 328.9 g l(-1) and 0.0333 h(-1) for a maximum Q of 2.73 mg l(-1) h(-1). The model of specific production rate (rho, mg INF-tau g(-1) cells h(-1)) was also established and showed the optimal Xc 287.7 g l(-1) and D=0.0361 h(-1) for the maximum rho (predicted to be 8.92 x 10(-3) mg(-1) g(-1) h(-1)). The methanol specific consumption rate (nu, g methanol g(-1) cells h(-1)) was calculated and shown to be independent of the cell density. The relationship between nu and mu (specific growth rate) was the same as that discovered from fed-batch fermentations of the same strain. The approach developed in this study is expected to be applicable to the optimization of continuous fermentations by other microorganisms.