title: Effects of nutrient levels and average culture pH on the glycosylation pattern of camelid-humanized monoclonal antibody.
abstract: The impact of operating conditions on the glycosylation pattern of humanized camelid monoclonal antibody, EG2-hFc produced by Chinese hamster ovary (CHO) cells has been evaluated by a combination of experiments and modeling. Cells were cultivated under different levels of glucose and glutamine concentrations with the goal of investigating the effect of nutrient depletion levels and ammonia build up on the cell growth and the glycoprofiles of the monoclonal antibody (Mab). The effect of average pH reduction on glycosylation level during the entire culture time or during a specific time span was also investigated. The relative abundance of glycan structures was quantified by hydrophilic interaction liquid chromatography (HILIC) and the galactosylation index (GI) and the sialylation index (SI) were determined. Lower initial concentrations of glutamine resulted in lower glucose consumption and lower cell yield but increased GI and SI levels when compared to cultures started with higher initial glutamine levels. Similarly, reducing the average pH of culture resulted in lower growth but higher SI and GI levels. These findings indicate that there is a tradeoff between cell growth, resulting Mab productivity and the achievement of desirable higher glycosylation levels. A dynamic model, based on a metabolic flux analysis (MFA), is proposed to describe the metabolism of nutrients, cell growth and Mab productivity. Finally, existing software (GLYCOVIS) that describes the glycosylation pathways was used to illustrate the impact of extracellular species on the glycoprofiles.
authors.affiliation: Chemical Engineering Department, University of Waterloo, Waterloo, ON, Canada. Electronic address: h3aghamo@uwaterloo.ca.
authors.forename: Hengameh
authors.initials: H
authors.lastname: Aghamohseni
authors.affiliation: Chemical Engineering Department, University of Waterloo, Waterloo, ON, Canada. Electronic address: skohadi@uwaterloo.ca.
authors.forename: Kaveh
authors.initials: K
authors.lastname: Ohadi
authors.affiliation: Microbiology Department, University of Manitoba, Winnipeg, MB, Canada. Electronic address: maureen.spearman@umanitoba.ca.
authors.forename: Maureen
authors.initials: M
authors.lastname: Spearman
authors.affiliation: Microbiology Department, University of Manitoba, Winnipeg, MB, Canada. Electronic address: umokunn@myumanitoba.ca.
authors.forename: Natalie
authors.initials: N
authors.lastname: Krahn
authors.affiliation: Chemical Engineering Department, University of Waterloo, Waterloo, ON, Canada. Electronic address: mooyoung@uwaterloo.ca.
authors.forename: Murray
authors.initials: M
authors.lastname: Moo-Young
authors.affiliation: Chemical Engineering Department, University of Waterloo, Waterloo, ON, Canada. Electronic address: jscharer@uwaterloo.ca.
authors.forename: Jeno M
authors.initials: JM
authors.lastname: Scharer
authors.affiliation: Microbiology Department, University of Manitoba, Winnipeg, MB, Canada. Electronic address: butler@cc.umanitoba.ca.
authors.forename: Mike
authors.initials: M
authors.lastname: Butler
authors.affiliation: Chemical Engineering Department, University of Waterloo, Waterloo, ON, Canada. Electronic address: hbudman@uwaterloo.ca.
authors.forename: Hector M
authors.initials: HM
authors.lastname: Budman
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry* / Q000378:metabolism*; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D005110:Extracellular Space / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D005973:Glutamine; D005947:Glucose
keywords: Dynamic modeling; Glycosylation; Glycosylation index; Mammalian cell culture; Monoclonal antibody