This review concerns the issues involved in the industrial development of fed-batch culture processes with Saccharomyces cerevisiae strains producing heterologous proteins. Most of process development considerations with fed-batch recombinant cultures are linked to the reliability and reproducibility of the process for manufacturing environments where quality assurance and quality control aspects are paramount. In this respect, the quality, safety and efficacy of complex biologically active molecules produced by recombinant techniques are strongly influenced by the genetic background of the host strain, genetic stability of the transformed strain and production process factors. An overview of the recent literature of these culture-related factors is coupled with our experience in yeast fed-batch process development for producing various therapeutic grade proteins. The discussion is based around three principal topics: genetics, microbial physiology and fed-batch process design. It includes the fundamental aspects of yeast strain physiology, the nature of the recombinant product, quality control aspects of the biological product, features of yeast expression vectors, expression and localization of recombinant products in transformed cells and fed-batch process considerations for the industrial production of Saccharomyces cerevisiae recombinant proteins. It is our purpose that this review will provide a comprehensive understanding of the fed-batch recombinant production processes and challenges commonly encountered during process development.

This review concerns the issues involved in the industrial development of fed-batch culture processes with Saccharomyces cerevisiae strains producing heterologous proteins. Most of process development considerations with fed-batch recombinant cultures are linked to the reliability and reproducibility of the process for manufacturing environments where quality assurance and quality control aspects are paramount. In this respect, the quality, safety and efficacy of complex biologically active molecules produced by recombinant techniques are strongly influenced by the genetic background of the host strain, genetic stability of the transformed strain and production process factors. An overview of the recent literature of these culture-related factors is coupled with our experience in yeast fed-batch process development for producing various therapeutic grade proteins. The discussion is based around three principal topics: genetics, microbial physiology and fed-batch process design. It includes the fundamental aspects of yeast strain physiology, the nature of the recombinant product, quality control aspects of the biological product, features of yeast expression vectors, expression and localization of recombinant products in transformed cells and fed-batch process considerations for the industrial production of Saccharomyces cerevisiae recombinant proteins. It is our purpose that this review will provide a comprehensive understanding of the fed-batch recombinant production processes and challenges commonly encountered during process development.

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High production levels of recombinant cholera toxin B subunit in Escherichia co/iwere obtained with the design of an efficient fed-batch process and control strategy. The fed-batch results demonstrated a biomass production of 58 g/L (Cell Dry Weight) attaining production levels of heterologous protein of 4.7g/L in the intracellular fraction, 0.96 g/L exported into the periplasm and 0.27 g/L secreted into the culture supernatant.

The recombinant outer-surface protein A with an N-terminally truncated form (des-Cys1-OspA) from the Lyme disease spirochete Borrelia burgdorferi was expressed in Saccharomyces cerevisiae at high production levels. Since the recombinant vaccine candidate expressed in Escherichia coli exhibits low production yields and the purification of lipoproteins appears to be difficult, we have investigated the secretion of a soluble recombinant OspA in the yeast S. cerevisiae. In this way, a Leu+ derivative of S. cerevisiae cI3ABYS86 was used as the host strain transformed with an expression plasmid containing the gene encoding des-Cys1-OspA and driven by the MF alpha 1 promoter. The fed-batch culture results revealed that an efficient secretion of des-Cys1-OspA is obtained with a high production level of about 2.1 g l-1 at a cell density of 101 g l-1 cell dry weight. The accumulation of recombinant protein in the supernatant exceeds 6% of the total yeast proteins when estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Moreover, des-Cys1-OspA showed lower solubilities at high cell densities and, as a consequence, a fraction of the recombinant protein precipitated. An internal cleavage of the MF alpha 1 pro::des-Cys1-OspA precursor was also detected. However, in this case the cleavage occurred at a frequency such that the large amounts of the secreted des-Cys1-OspA could be employed for the evaluation of an immunogenic effect on animal immunization. These studies will extend the knowledge of the usefulness of OspA as a vaccine for Lyme borreliosis.