title: Characterization of the acidic species of a monoclonal antibody using weak cation exchange chromatography and LC-MS.
abstract: Charge variants, especially acidic charge variants, of recombinant monoclonal antibodies have been challenging to fully characterize despite the fact that several posttranslational modifications have already been identified. The acidic species of a recombinant monoclonal antibody were collected using weak cation exchange (WCX)-10 chromatography and characterized by LC-MS at multiple levels. In this study, methionine oxidation and asparagine deamidation are the only two modifications identified in the acidic species. Incubation of the collected main chromatographic peak with hydrogen peroxide generated acidic species, which confirmed that acidic species were enriched in oxidized antibody. Differences observed between the original acidic species and the oxidization-induced acidic species indicate that different mechanisms are involved in the formation of acidic species. Additionally, acidic species were generated by thermal stress of the collected main peak from the original sample. Thermal stress of the collected main peak in pH 9 buffer or ammonium bicarbonate generated chromatograms that are highly similar to those from the analysis of the original molecule. LC-MS analysis identified oxidation of the same methionine residue and deamidation of the same asparagine in the corresponding acidic fractions generated by thermal stress; however, relatively lower levels of methionine oxidation and higher levels of asparagine deamdiation were observed. The results support the use of stressed conditions to generate low abundance species for detailed characterization of recombinant monoclonal antibody charge variants, but with caution.
authors.affiliation: Product Characterization and ‡Biochemical Process Development, Alexion Pharmaceuticals Inc. , 352 Knotter Drive, Cheshire, Connecticut, 06410, United States.
authors.forename: Gomathinayagam
authors.initials: G
authors.lastname: Ponniah
authors.affiliation: Product Characterization and ‡Biochemical Process Development, Alexion Pharmaceuticals Inc. , 352 Knotter Drive, Cheshire, Connecticut, 06410, United States.
authors.forename: Adriana
authors.initials: A
authors.lastname: Kita
authors.affiliation: Product Characterization and ‡Biochemical Process Development, Alexion Pharmaceuticals Inc. , 352 Knotter Drive, Cheshire, Connecticut, 06410, United States.
authors.forename: Christine
authors.initials: C
authors.lastname: Nowak
authors.affiliation: Product Characterization and ‡Biochemical Process Development, Alexion Pharmaceuticals Inc. , 352 Knotter Drive, Cheshire, Connecticut, 06410, United States.
authors.forename: Alyssa
authors.initials: A
authors.lastname: Neill
authors.affiliation: Product Characterization and ‡Biochemical Process Development, Alexion Pharmaceuticals Inc. , 352 Knotter Drive, Cheshire, Connecticut, 06410, United States.
authors.forename: Yekaterina
authors.initials: Y
authors.lastname: Kori
authors.affiliation: Product Characterization and ‡Biochemical Process Development, Alexion Pharmaceuticals Inc. , 352 Knotter Drive, Cheshire, Connecticut, 06410, United States.
authors.forename: Saravanamoorthy
authors.initials: S
authors.lastname: Rajendran
authors.affiliation: Product Characterization and ‡Biochemical Process Development, 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 / Q000737:chemistry*; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003412:Cricetulus; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry*; D013058:Mass Spectrometry; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011994:Recombinant Proteins