title: Characterization of charge variants of a monoclonal antibody using weak anion exchange chromatography at subunit levels.
abstract: An efficient strategy to characterize recombinant monoclonal antibody charge variants was established using weak anion exchange chromatography, LC-MS and IdeS digestion to allow subunit level characterization. Significantly higher resolution was achieved at subunit levels by weak anion exchange chromatography and LC-MS. In addition, subunit analysis localized potential modifications to either F(ab')2or Fc fragments to facilitate further characterization. Peptide mapping of fractions from various charge variants after IdeS digestion identified aspartate isomerization, asparagine deamidation and glycation as the modifications. Although, aspartate isomerization does not generate net charge difference directly, it does generate antibody basic species. Antibodies with either isoaspartate or aspartate from deamidation showed different retention times by chromatography. Even more interestingly, the antibody contained succinimide as the isomerization intermediate, which though more basic compared to aspartate, eluted off the weak anion exchange column as an acidic species. The results demonstrated not only the utility of subunit level characterization but also the unpredictable chromatographic behavior of antibody charge variants.
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, 100 College Street, New Haven, CT06510, United States.
authors.forename: Gomathinayagam
authors.initials: G
authors.lastname: Ponniah
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, 100 College Street, New Haven, CT06510, United States.
authors.forename: Christine
authors.initials: C
authors.lastname: Nowak
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, 100 College Street, New Haven, CT06510, United States.
authors.forename: Alyssa
authors.initials: A
authors.lastname: Neill
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, 100 College Street, New Haven, CT06510, United States. Electronic address: LiuH@alxn.com.
authors.forename: Hongcheng
authors.initials: H
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics; D007536:Isomerism; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D010455:Peptides; D011994:Recombinant Proteins
keywords: LC-MS; Posttranslational modifications; Recombinant monoclonal antibodies; Succinimide