title: Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry.
abstract: MAb1, a human IgG1 monoclonal antibody produced in a NS0 cell line, exhibits charge heterogeneity because of the presence of variants formed by processes such as N-terminal glutamate cyclization, C-terminal lysine truncation, deamidation, aspartate isomerization and sialylation in the carbohydrate moiety. Four major charge variants of MAb1 were isolated and the conformations of these charge variants were studied using hydrogen/deuterium exchange mass spectrometry, including the H/D exchange time course (HX-MS) and the stability of unpurified proteins from rates of H/D exchange (SUPREX) techniques. HX-MS was used to evaluate the conformation and solution dynamics of MAb1 charge variants by measuring their deuterium buildup over time at the peptide level. The SUPREX technique evaluated the unfolding profile and relative stability of the charge variants by measuring the exchange properties of globally protected amide protons in the presence of a chemical denaturant. The H/D exchange profiles from both techniques were compared among the four charge variants of MAb1. The two techniques together offered extensive understanding about the local and subglobal/global unfolding of the charge variants of MAb1. Our results demonstrated that all four charge variants of MAb1 were not significantly different in conformation, solution dynamics and chemical denaturant-induced unfolding profile and stability, which aids in understanding the biofunctions of the molecules. The analytical strategy used for conformational characterization may also be applicable to comparability studies done for antibody therapeutics.
authors.affiliation: BioAnalytical Sciences, ImClone Systems Corporation, Wholly-Owned Subsidiary of Eli Lilly and Company, Branchburg, NJ, USA.
authors.forename: Liangjie
authors.initials: L
authors.lastname: Tang
authors.forename: Shanmuuga
authors.initials: S
authors.lastname: Sundaram
authors.forename: Jingming
authors.initials: J
authors.lastname: Zhang
authors.forename: Ping
authors.initials: P
authors.lastname: Carlson
authors.forename: Alice
authors.initials: A
authors.lastname: Matathia
authors.forename: Babita
authors.initials: B
authors.lastname: Parekh
authors.forename: Qinwei
authors.initials: Q
authors.lastname: Zhou
authors.forename: Ming-Ching
authors.initials: MC
authors.lastname: Hsieh
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D003903:Deuterium / Q000032:analysis*; D006801:Humans; D006859:Hydrogen / Q000032:analysis*; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D010449:Peptide Mapping; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D058767:Protein Unfolding
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D006859:Hydrogen; D003903:Deuterium