Evaluation of different preparation parameters for the production and cryopreservation of seed cultures with recombinant Saccharomyces cerevisae.

In this article we describe the intensive investigation of defined physicochemical parameters for their influence on the preparation, storage stability in liquid nitrogen, and the post-thaw productivity of a recombinant strain of Saccharomyces cerevisiae expressing the human factor XIIIa protein. Preparation of industrially sized seed cultures and their storage stability were monitored over more than a 1-year period. Major parameters recorded before and after thawing were number of colony-forming units on agar slant media, plasmid retention, and expression capacity of the recombinant protein. The viability of the cells after reconstitution of the frozen batches was found to improve significantly if a certain combination of prethaw growth conditions was applied and amino acids were included in the cryoprotectant media. In addition, the influence of different concentrations of glycerol as a cryoprotectant was investigated and the specific freezing conditions which improved the viability were identified. Even without complex freezing devices for the controlled freezing of cultures, viability rates of 85% and higher were obtained, enabling consistent production of the recombinant protein.