title: Assessing the viability of a clumpy mnn9 strain of Saccharomyces cerevisiae used in the manufacture of recombinant pharmaceutical proteins.
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.
authors.affiliation: Department of Bioprocess and Bioanalytical Research, Merck Research Laboratories, WP17-101, Sumneytown Pike, West Point, PA 19486, USA. paul_duncan@merck.com
authors.forename: Paul A
authors.initials: PA
authors.lastname: Duncan
authors.forename: Sean
authors.initials: S
authors.lastname: Gallagher
authors.forename: Linda
authors.initials: L
authors.lastname: McKerral
authors.forename: P K
authors.initials: PK
authors.lastname: Tsai
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002470:Cell Survival; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D013194:Staining and Labeling; D013696:Temperature
chemical_list: D011994:Recombinant Proteins