title: A low redox potential affects monoclonal antibody assembly and glycosylation in cell culture.
abstract: Glycosylation and intracellular assembly of monoclonal antibodies (MAbs) is important for glycan profile consistency. To better understand how these factors may be influenced by a lower redox potential, an IgG1-producing NS0 cell line was grown in the presence of varying concentrations of dithiothreitol (DTT). Cultures were monitored for growth and culture redox potential (CRP) with glycan heterogeneity determined using a HILIC-HPLC method. Macroheterogeneity was unchanged in all conditions whereas the Galactosylation Index (GI) decreased by as much as 50% in cultures with lower CRP or higher dithiothreitol levels. This shift in GI is reflected in more agalactosylated and asialylated species being produced. The MAb assembly pathway was determined using radioactive isotope35S incorporated into nascent IgG1 molecules. The assembly pathway for this IgG1 was shown to progress via HC→HC2→HC2LC→HC2LC2in all conditions tested and autoradiographs highlighted that the ratio of heavy chain dimer to heavy chain monomer increased over time with increasing DTT concentrations. This increase and correspondingly lower GI values may be due to disruption of the disulfide bonds at higher levels of assembly. A change in the assembly pathway may alter the final IgG glycan pattern and lead to control mechanisms that influence glycan profiles of MAbs.
authors.affiliation: University of Manitoba, Department of Microbiology, Winnipeg R3T 2N2, Manitoba, Canada.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Dionne
authors.affiliation: University of Manitoba, Department of Microbiology, Winnipeg R3T 2N2, Manitoba, Canada.
authors.forename: Neha
authors.initials: N
authors.lastname: Mishra
authors.affiliation: University of Manitoba, Department of Microbiology, Winnipeg R3T 2N2, Manitoba, Canada. Electronic address: Michael.Butler@umanitoba.ca.
authors.forename: Michael
authors.initials: M
authors.lastname: Butler
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004229:Dithiothreitol / Q000494:pharmacology*; D006031:Glycosylation / Q000187:drug effects; D051379:Mice; D010084:Oxidation-Reduction / Q000187:drug effects
chemical_list: D000911:Antibodies, Monoclonal; D004229:Dithiothreitol
keywords: Glycosylation; HILIC-HPLC; Monoclonal antibody; NS0; Redox potential