title: Characterization of Recombinant Monoclonal Antibody Charge Variants Using OFFGEL Fractionation, Weak Anion Exchange Chromatography, and Mass Spectrometry.
abstract: Recombinant monoclonal antibody charge heterogeneity has been commonly observed as multiple bands or peaks when analyzed by charge-based analytical methods such as isoelectric focusing electrophoresis and cation or anion exchange chromatography. Those charge variants have been separated by some of the above-mentioned methods and used for detailed characterization. The utility of a combination of OFFGEL fractionation and weak anion exchange chromatography to separate the charge variants of a recombinant monoclonal antibody was demonstrated in the current study. Charge variants were separated into various fractions of high purity and then analyzed thoroughly by liquid chromatography mass spectrometry. Analysis of intact molecular weights identified the presence of heavy chain leader sequence, C-terminal lysine, and C-terminal amidation. The identified modifications were further localized into different regions of the antibody from analysis of antibody fragments obtained from FabRICATOR digestion. Analysis of tryptic peptides from various fractions further confirmed the previously identified modifications in the basic variants. Asparagine deamidation and aspartate isomerization were identified in acidic fractions from analysis of tryptic peptides. Basic variants have been fully accounted for by the identified modifications. However, only a portion of the acidic variants can be explained by deamidation and isomerization, suggesting that additional modifications are yet to be identified or acidic variants are an ensemble of molecules with different structures.
authors.affiliation: Product Characterization, Alexion Pharmaceuticals Inc, 352 Knotter Drive, Cheshire, Connecticut 06410, United States.
authors.forename: Alyssa
authors.initials: A
authors.lastname: Neill
authors.affiliation: Product Characterization, Alexion Pharmaceuticals Inc, 352 Knotter Drive, Cheshire, Connecticut 06410, United States.
authors.forename: Christine
authors.initials: C
authors.lastname: Nowak
authors.affiliation: Product Characterization, Alexion Pharmaceuticals Inc, 352 Knotter Drive, Cheshire, Connecticut 06410, United States.
authors.forename: Rekha
authors.initials: R
authors.lastname: Patel
authors.affiliation: Product Characterization, Alexion Pharmaceuticals Inc, 352 Knotter Drive, Cheshire, Connecticut 06410, United States.
authors.forename: Gomathinayagam
authors.initials: G
authors.lastname: Ponniah
authors.affiliation: Product Characterization, Alexion Pharmaceuticals Inc, 352 Knotter Drive, Cheshire, Connecticut 06410, United States.
authors.forename: Nidia
authors.initials: N
authors.lastname: Gonzalez
authors.affiliation: Product Characterization, Alexion Pharmaceuticals Inc, 352 Knotter Drive, Cheshire, Connecticut 06410, United States.
authors.forename: Dino
authors.initials: D
authors.lastname: Miano
authors.affiliation: Product Characterization, Alexion Pharmaceuticals Inc, 352 Knotter Drive, Cheshire, Connecticut 06410, United States.
authors.forename: Hongcheng
authors.initials: H
authors.lastname: Liu
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D016466:CHO Cells; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003412:Cricetulus; D013058:Mass Spectrometry; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000032:analysis
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins