Analysis of post-translational modifications in recombinant monoclonal antibody IgG1 by reversed-phase liquid chromatography/mass spectrometry.
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.
Yan
Boxu
B
Department of Analytical Science, Amgen Inc, Seattle, WA 98119, USA. byan@amgen.com
Valliere-Douglass
John
J
Brady
Lowell
L
Steen
Sean
S
Han
Mei
M
Pace
Danielle
D
Elliott
Susan
S
Yates
Zac
Z
Han
Yihong
Y
Balland
Alain
A
Wang
Weichun
W
Pettit
Dean
D
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
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D014357:Trypsin; D010206:Papain