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

Abstract

This paper provides an approach for optimizing the cell density (X_c) and dilution rate (D) in a chemostat for a Pichia pastoris continuous fermentation for the extracellular production of a recombinant protein, interferon τ (INF-τ). The objective was to maximize the volumetric productivity (Q, mg INF-τ l⁻¹ h⁻¹), which was accomplished using response surface methodology (RSM) to model the response of Q as a function of X_c and D within the ranges 150≤ X_c ≤450 g cells (wet weight) l⁻¹ and 0.1 μ_m≤D≤0.9 μ_m (μ_m=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 X_c and D were determined based on the mass balance. From the RSM model, the optimal X_c 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 (ρ, mg INF-τ g⁻¹ cells h⁻¹) was also established and showed the optimal X_c=287.7 g l⁻¹ and D=0.0361 h⁻¹ for the maximum ρ (predicted to be 8.92×10⁻³ mg⁻¹ g⁻¹ h⁻¹). The methanol specific consumption rate (ν, g methanol g⁻¹ cells h⁻¹) was calculated and shown to be independent of the cell density. The relationship between ν and μ (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.