Optimization of Specific Product Formation Rate by Statistical and Formal Kinetic Model Descriptions of an HSA Producing Pichia pastoris Muts Strain

B. Kupcsulik and B. Sevella

Abstract
Pichia pastoris has proven to be an ideal host for eukaryotic protein expression. Numerous models have been developed to describe the heterologous product formation of this yeast, however the pH and temperature dependence has not been characterized yet. A linear full factorial design with a centre point was expanded with a second order Box-Behnken design towards increasing product yields to examine the influence of pH and temperature on the product formation of a human serum albumin (HSA) producing P. pastoris GS115 MutS strain in methanol-induced fermentations. Both the linear and quadratic form of pH had significant effects on the product yield, productivity and specific product formation rate, whereas the effect of temperature was less important. As the specific growth rate was independent of pH and temperature in the product forming experimental range, the volumetric productivity was determined mainly by the specific product formation rate. For this reason, the optimization of productivity requires the growth independent description of specific product formation rate. Beside a parabolic statistical model, an empirically based formula was fitted to the experimental results in order to characterize product formation. The empirical formula was composed of three separate terms i.e. the specific maximal product formation rate, a substrate inhibition kinetics module for [H+] and two Arrhenius equations describing the temperature dependence of product formation. The maximum specific product formation rate was measured at the optimum, predicted by the empirical formula (pH5.64and 20.24°C), which slightly exceeded the calculated value (0.370 mg HSA per g DW and h vs. 0.354mg HSA per g DW and h).

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Keywords
Pichia pastoris, product formation, model, pH dependence, temperature dependence