title: Analysis of post-translational modifications in recombinant monoclonal antibody IgG1 by reversed-phase liquid chromatography/mass spectrometry.
abstract: Characterization and quantitative analysis of modifications in recombinant monoclonal antibodies (mAbs) plays an important role in biopharmaceutical development. This study demonstrates a new approach to assess variants in mAbs, based on individual analysis of subdomains. These subdomains were generated by dithiothreitol reduction and papain cleavage. A reversed-phase LC-MS method was developed that provides efficient separation of subdomains (light and heavy chains, Fab and Fc) containing several specific modifications such as pyroglutamic acid, deamidation, isomerization and oxidation. The best separation was achieved on Zorbax SB C8 columns using linear water-acetonitrile gradients in 0.1% trifluoroacetic acid. Deconvoluted electrospray ionization mass spectra of these domains revealed the modification profiles of these variants with high accuracy and resolution. This study presents a strategy that offers orthogonal approaches to analyze antibody variants, and provide a qualitative and quantitative assessment of mAb heterogeneity.
authors.affiliation: Department of Analytical Science, Amgen Inc, Seattle, WA 98119, USA. byan@amgen.com
authors.forename: Boxu
authors.initials: B
authors.lastname: Yan
authors.forename: John
authors.initials: J
authors.lastname: Valliere-Douglass
authors.forename: Lowell
authors.initials: L
authors.lastname: Brady
authors.forename: Sean
authors.initials: S
authors.lastname: Steen
authors.forename: Mei
authors.initials: M
authors.lastname: Han
authors.forename: Danielle
authors.initials: D
authors.lastname: Pace
authors.forename: Susan
authors.initials: S
authors.lastname: Elliott
authors.forename: Zac
authors.initials: Z
authors.lastname: Yates
authors.forename: Yihong
authors.initials: Y
authors.lastname: Han
authors.forename: Alain
authors.initials: A
authors.lastname: Balland
authors.forename: Weichun
authors.initials: W
authors.lastname: Wang
authors.forename: Dean
authors.initials: D
authors.lastname: Pettit
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D007074:Immunoglobulin G / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010206:Papain / Q000378:metabolism; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D014357:Trypsin; D010206:Papain