A rational approach to improving productivity in recombinant Pichia pastoris fermentation

A Mut(S) Pichia pastoris strain that had been genetically modified to produ ce and secrete sea raven antifreeze protein was used as a model system to d emonstrate the implementation of a rational, model-based approach to improv e process productivity. A set of glycerol/methanol mixed-feed continuous st irred-tank reactor (CSTR) experiments was performed at the 5-L scale to cha racterize the relationship between the specific growth rate and the cell yi eld on methanol, the specific methanol consumption rate, the specific recom binant protein formation rate, and the productivity based on secreted prote in levels. The range of dilution rates studied was 0.01 to 0.10 h(-1). and the residual methanol concentration was kept constant at approximately 2 g/ L (below the inhibitory level). With the assumption that the cell yield on glycerol was constant, the cell yield on methanol increased from approximat ely 0.5 to 1.5 over the range studied. A maximum specific methanol consumpt ion rate of 20 mg/g.h was achieved at a dilution rate of 0.06 h(-1). The sp ecific product formation rate and the volumetric productivity based on prod uct continued to increase over the range of dilution rates studied, and the maximum values were 0.06 mg/g.h and 1.7 mg/L.h, respectively. Therefore, n o evidence of repression by glycerol was observed over this range, and oper ating at the highest dilution rate studied maximized productivity. Fed-batc h mass balance equations, based on Monod-type kinetics and parameters deriv ed from data collected during the CSTR work, were then used to predict cell growth and recombinant protein production and to develop an exponential fe eding strategy using two carbon sources. Two exponential fed-batch fermenta tions were conducted according to the predicted feeding strategy at specifi c growth rates of 0.03 h(-1) and 0.07 h(-1) to verify the accuracy of the m odel. Cell growth was accurately predicted in both fed-batch runs; however, the model underestimated recombinant product concentration. The overall vo lumetric productivity of both runs was approximately 2.2 mg/L.h, representi ng a tenfold increase in the productivity compared with a heuristic feeding strategy. (C) 2001 John Wiley & Sons, Inc.