Assessing the viability of a clumpy mnn9 strain of Saccharomyces cerevisiae used in the manufacture of recombinant pharmaceutical proteins

J Ind Microbiol Biotechnol. 2004 Dec;31(11):500-6. doi: 10.1007/s10295-004-0177-y. Epub 2004 Oct 26.

Abstract

Demonstration of the viability of cryopreserved cell bank used to make a biopharmaceutical product is an important indicator of the ability to consistently manufacture over a long period of time, and is mandated in regulatory guidances. A mnn9 strain of Saccharomyces cerevisiae, chosen for its inability to hypermannosylate vaccine antigens, has a clumpy growth tendency due to the inactivation of the gene MNN9 (wild-type), complicating the interpretation of conventional viability measurements useful for single cells. Therefore, two growth-based measurements as well as staining by a membrane-impermeable dye were examined for their ability to reflect changes in viability of a clumpy mnn9 (defective) strain. The cell clumps proved to be stable to mixing, and variability of agar-plate-based viable counts (VC) of undisrupted suspensions of this clumpy mnn9 strain was consistent with variability observed for cell banks of a non-clumpy MNN9 strain. Both the VC and the growth times in an oxygen-sensing broth-based microplate assay corresponded well with shake-flask growth times for a set of stressed and unstressed samples, although the correlation was highest between the two broth-based systems. Counts of trypan-blue-stained cells within clumps also increased with time of stress, suggesting that this method could be adapted as a simple index of viability as well.

MeSH terms

  • Biotechnology / methods*
  • Cell Survival
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / pharmacology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / growth & development*
  • Saccharomyces cerevisiae / metabolism
  • Staining and Labeling
  • Temperature

Substances

  • Recombinant Proteins