A Rational Approach to Improving Productivity in Recombinant Pichia pastoris Fermentation Marc C. d’Anjou, Andrew J. Daugulis Department of Chemical Engineering, Queen’s University, Kingston, Ontario K7L 3N6, Canada; telephone: 613-533-2784; fax: 613-533-6637; e-mail: daugulis@chee.queensu.ca Received 13 February 2000; accepted 22 June 2000 Abstract: A Mut S Pichia pastoris strain that had been genetically modified to produce and secrete sea raven antifreeze protein was used as a model system to dem- onstrate the implementation of a rational, model-based approach to improve process productivity. A set of glyc- erol/methanol mixed-feed continuous stirred-tank reac- tor (CSTR) experiments was performed at the 5-L scale to characterize the relationship between the specific growth rate and the cell yield on methanol, the specific methanol consumption rate, the specific recombinant protein for- mation rate, and the productivity based on secreted pro- tein 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 inhibi- tory level). With the assumption that the cell yield on glycerol was constant, the cell yield on methanol in- creased from approximately 0.5 to 1.5 over the range studied. A maximum specific methanol consumption rate of 20 mg/g ? h was achieved at a dilution rate of 0.06 h -1 . The specific product formation rate and the volumet- ric productivity based on product continued to increase over the range of dilution rates studied, and the maxi- mum values were 0.06 mg/g ? h and 1.7 mg/L ? h, respec- tively. Therefore, no evidence of repression by glycerol was observed over this range, and operating at the high- est dilution rate studied maximized productivity. Fed- batch mass balance equations, based on Monod-type ki- netics and parameters derived from data collected dur- ing the CSTR work, were then used to predict cell growth and recombinant protein production and to develop an exponential feeding strategy using two carbon sources. Two exponential fed-batch fermentations were con- ducted according to the predicted feeding strategy at specific growth rates of 0.03 h -1 and 0.07 h -1 to verify the accuracy of the model. Cell growth was accurately pre- dicted in both fed-batch runs; however, the model under- estimated recombinant product concentration. The over- all volumetric productivity of both runs was approxi- mately 2.2 mg/L ? h, representing a tenfold increase in the productivity compared with a heuristic feeding strat- egy. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 1–11, 2001. Keywords: Pichia pastoris; recombinant proteins; fed- batch; fermentation INTRODUCTION Pichia pastoris, an ascosporogenic, methylotrophic yeast, is finding increasing use as a host for the expression of a wide variety of recombinant proteins at the bench and pilot scales. There are a number of excellent reviews describing the general features of this yeast expression system (Cregg and Higgins, 1995; Faber et al., 1995; Romanos et al., 1992; Vedvick, 1991) and examining recent advances in its de- velopment and application (Cregg et al., 1993; Hollenberg and Gellissen, 1997; Romanos, 1995; Sreekrishna et al., 1997). At first glance, Pichia seems to be an ideal host, because: (i) it is a simple microbe that is capable of growth to high cell densities on inexpensive, defined media using well-developed fermentation protocols; (ii) expression of foreign proteins in Pichia is driven by the very strong and tightly regulated AOX1 promoter that has been exploited by a number of vectors with reasonable transformation effi- ciencies showing stable chromosomal integration; (iii) as simple eukaryotes, yeasts are capable of many of the same posttranslational modifications to proteins as higher ani- mals; and (iv) yeasts are capable of secreting high levels of many proteins, simplifying downstream purification. How- ever, simply inserting a gene of interest into a vector and transforming a microbial host is no guarantee of a viable bioprocess. Expression levels reported in the literature for foreign proteins produced in Pichia are highly variable and range from the milligrams-per-liter to grams-per-liter levels. The expression level for a given recombinant protein pro- duced in Pichia seems to be decided largely by its inherent properties such as the amino acid sequence, the tertiary structure, and the site of expression (Sreekrishna et al., 1997). There are a number of approaches that can be taken to improve this critical parameter; however, many of these are empirical by nature, and their reporting is anecdotal. Another strategy, which has been applied successfully in more developed recombinant expression systems, such as Escherichia coli and Saccharomyces cerevisiae (Baheri et al., 1997; Hardjito et al., 1992), involves taking a system- atic, rational approach to optimizing the entire process. This strategy involves initiating a study designed to identify and to characterize trends in the behavior of the system. These Correspondence to: A. Daugulis Contract grant sponsors: Lloyd Carr-Harris Foundation; Natural Sci- ences and Engineering Research Council of Canada © 2001 John Wiley & Sons, Inc.