title: Components of yeast (Sacchromyces cervisiae) extract as defined media additives that support the growth and productivity of CHO cells.
abstract: Yeast and plant hydrolysates are used as media supplements to support the growth and productivity of CHO cultures for biopharmaceutical production. Through fractionation of a yeast lysate and metabolic analysis of a fraction that had bioactivity equivalent to commercial yeast extract (YE), bioactive components were identified that promoted growth and productivity of two recombinant CHO cell lines (CHO-Luc and CHO-hFcEG2) equivalent to or greater than YE-supplemented media. Autolysis of the yeast lysate was not necessary for full activity, suggesting that the active components are present in untreated yeast cells. A bioactive fraction (3KF) of the yeast lysate was isolated from the permeate using a 3kDa molecular weight cut-off (MWCO) filter. Supplementation of this 3KF fraction into the base media supported growth of CHO-Luc cells over eight passages equivalent to YE-supplemented media. The 3KF fraction was fractionated further by a cation exchange spin column using a stepwise pH elution. Metabolomic analysis of a bioactive fraction isolated at high pH identified several arginine and lysine-containing peptides as well as two polyamines, spermine and spermidine, with 3.5× and 4.5× higher levels compared to a fraction showing no bioactivity. The addition of a mixture of polyamines and their precursors (putrescine, spermine, spermidine, ornithine and citrulline) as well as increasing the concentration of some of the components of the original base medium resulted in a chemically-defined (CD) formulation that produced an equivalent viable cell density (VCD) and productivity of the CHO-Luc cells as the YE-supplemented medium. The VCD of the CHO-hFcEG2 culture in the CD medium was 1.9× greater and with equivalent productivity to the YE-supplemented media.
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada.
authors.forename: Maureen
authors.initials: M
authors.lastname: Spearman
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada.
authors.forename: Sarah
authors.initials: S
authors.lastname: Chan
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada.
authors.forename: Vince
authors.initials: V
authors.lastname: Jung
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Kowbel
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada.
authors.forename: Meg
authors.initials: M
authors.lastname: Mendoza
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada.
authors.forename: Vivian
authors.initials: V
authors.lastname: Miranda
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada. Electronic address: butler@umanitoba.ca.
authors.forename: Michael
authors.initials: M
authors.lastname: Butler
mesh_terms: D000818:Animals; D001688:Biological Products / Q000737:chemistry / Q000494:pharmacology; D016466:CHO Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001688:Biological Products; D017077:Culture Media, Conditioned
keywords: Antibody; CHO; Fractionation; Hydrolysate; Polyamines; Yeast