Recombinant Protein Production with Pichia pastoris in Continuous Fermentation – Kinetic Analysis of Growth and Product Formation

Abstract

Continuous fermentation was applied to the production of recombinant human chymotrypsinogen B (hCTRB) by the methylotrophic yeast Pichia pastoris as a tool for the kinetic analysis of growth and product formation. Using methanol as the sole source of carbon, energy, and induction, cell growth could be described by a non-competitive Monod approach. Maximum growth rate μmax was determined to 0.084 h--1 and the KM-value for methanol to 0.22 g·L--1, respectively. With respect to product formation, a similar model was established exhibiting a methanol concentration of 0.13 g·L--1 as the KM-value and a maximum biomass-specific product-formation rate of πmax = 0.23 mg·g--1·h--1. The production of hCTRB was strictly growth-coupled. The data provided covers the range of methanol concentrations between 0 and 4 g·L--1. Substrate concentrations exceeding this upper value led to a complete collapse of product formation. This change in phenotype turned out to be irreversible indicating a genetic instability of transformed Pichia pastoris caused by excess methanol.