title: The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody.
abstract: The glycosylation pattern of a chimeric heavy chain antibody (EG2) produced from CHO cells was affected by the glucose concentration (0-25mM) of cultures established at high density (>10(6)ml(-1)) over 24h. The resulting proportion of non-glycosylated Mab was directly correlated to the exposure time of cells to media depleted of glucose. Deprivation of glucose for the full 24h resulted in a 45% non-glycosylated Mab fraction. Analysis of steady state levels of intracellular lipid-linked oligosaccharides (LLOs) showed that under glucose limitation there was a reduction in the amount of full length LLO (Glc3Man9GlcNac2), with a concomitant increase in the smaller mannosyl-glycans (Man2-5GlcNAc2). Glycan microheterogeneity was quantified by galactosylation and sialylation indices (GI and SI) which showed a direct correlation to the cell specific glucose uptake. The GI increased to 0.83 following media supplementation with a cocktail of uridine, manganese and galactose. This is significantly higher than for a fully humanized antibody (DP12) produced under the similar conditions or for similar antibodies reported in the literature. The high GI of the chimeric antibody (EG2) may be due to its low molecular weight and unusual structure. These findings are important in relation to the low substrate that may occur in fed-batch cultures for Mab production.
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2.
authors.forename: Bo
authors.initials: B
authors.lastname: Liu
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2.
authors.forename: Maureen
authors.initials: M
authors.lastname: Spearman
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2.
authors.forename: John
authors.initials: J
authors.lastname: Doering
authors.affiliation: Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2.
authors.forename: Erica
authors.initials: E
authors.lastname: Lattová
authors.affiliation: Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2.
authors.forename: Hélène
authors.initials: H
authors.lastname: Perreault
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2. Electronic address: butler@cc.umanitoba.ca.
authors.forename: Michael
authors.initials: M
authors.lastname: Butler
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D016466:CHO Cells; D003412:Cricetulus; D003470:Culture Media; D005690:Galactose; D005947:Glucose / Q000378:metabolism*; D006031:Glycosylation; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D003470:Culture Media; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; C023023:lipid-linked oligosaccharides; D005947:Glucose; D005690:Galactose
keywords: CHO cells; Glycosylation; Lipid-linked oligosaccharide; Monoclonal antibody