title: Tuning a MAb glycan profile in cell culture: Supplementing N-acetylglucosamine to favour G0 glycans without compromising productivity and cell growth.
abstract: Glycosylation is a critical quality attribute of many therapeutic proteins, particularly monoclonal antibodies (MAbs). Nucleotide-sugar precursors supplemented to growth medium to affect the substrate supply chain of glycosylation has yielded promising but varied results for affecting glycosylation. Glucosamine (GlcN), a precursor for N-acetylglucosamine (GlcNAc), is a major component of mammalian glycans. The supplementation of GlcN to CHO cells stably-expressing a chimeric heavy-chain monoclonal antibody, EG2-hFc, reduces the complexity of glycans to favour G0 glycoforms, while also negatively impacting cell growth. Although several researchers have examined the supplementation of glucosamine, no clear explanation of its impact on cell growth has been forthcoming. In this work, the glucosamine metabolism is examined. We identified the acetylation of GlcN to produce GlcNAc to be the most likely cause for the negative impact on growth due to the depletion of intracellular acetyl-CoA pools in the cytosol. By supplementing GlcNAc in lieu of GlcN to CHO cells producing EG2-hFc, we achieve the same shift in glycan complexity with marginal impacts on the cell growth and protein production.
authors.affiliation: Waterloo Institute for Nanotechnology, Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario N2L 3G, Canada.
authors.forename: Eric J M
authors.initials: EJ
authors.lastname: Blondeel
authors.affiliation: Department of Microbiology, University of Manitoba, 79 Freedman Crescent, Fort Garry Campus, Winnipeg, MB R3T 2N2, Canada.
authors.forename: Katrin
authors.initials: K
authors.lastname: Braasch
authors.affiliation: Waterloo Institute for Nanotechnology, Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario N2L 3G, Canada.
authors.forename: Thomas
authors.initials: T
authors.lastname: McGill
authors.affiliation: Chenomx Inc., Edmonton, Alberta T5J 4P6, Canada.
authors.forename: David
authors.initials: D
authors.lastname: Chang
authors.affiliation: Waterloo Institute for Nanotechnology, Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario N2L 3G, Canada.
authors.forename: Christina
authors.initials: C
authors.lastname: Engel
authors.affiliation: Department of Microbiology, University of Manitoba, 79 Freedman Crescent, Fort Garry Campus, Winnipeg, MB R3T 2N2, Canada.
authors.forename: Maureen
authors.initials: M
authors.lastname: Spearman
authors.affiliation: Department of Microbiology, University of Manitoba, 79 Freedman Crescent, Fort Garry Campus, Winnipeg, MB R3T 2N2, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Butler
authors.affiliation: Waterloo Institute for Nanotechnology, Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario N2L 3G, Canada. Electronic address: marc.aucoin@uwaterloo.ca.
authors.forename: Marc G
authors.initials: MG
authors.lastname: Aucoin
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism*; D005944:Glucosamine / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D006031:Glycosylation; D009711:Nucleotides / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D003470:Culture Media; D009711:Nucleotides; D005947:Glucose; D005944:Glucosamine; D000117:Acetylglucosamine
keywords: CHO cells; Glucosamine GlcN; Glycosylation; N-acetylglucosamine GlcNAc; Nucleotide sugars; Protein quality