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

• Published in: Journal of industrial microbiology & biotechnology Vol. 31; no. 11; pp. 500 - 506
• Main Authors: DUNCAN, Paul A, GALLAGHER, Sean, MCKERRAL, Linda, TSAI, P. K
• Format: Journal Article
• Language: English
• Published: Heidelberg Springer 01-12-2004; Oxford University Press
• Subjects: Bacteria; Biological and medical sciences; Biotechnology; Biotechnology - methods; Cell Survival; Fundamental and applied biological sciences. Psychology; Inactivation; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae - metabolism; Staining and Labeling; Temperature; Yeast; Fungi; Yeast; Stability; Cell bank; Ascomycetes; Recombinant protein; Saccharomyces cerevisiae; Viability; Thallophyta; mnn9
• ISSN: 1367-5435; 1476-5535
• DOI: 10.1007/s10295-004-0177-y

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.