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Analysis of post-translational modifications in recombinant monoclonal antibody IgG1 by reversed-phase liquid chromatography/mass spectrometry

2007; Elsevier BV; Volume: 1164; Issue: 1-2 Linguagem: Inglês

10.1016/j.chroma.2007.06.063

1873-3778

Boxu Yan, John Valliere‐Douglass, Lowell J. Brady, Sean Steen, Mei Han, Danielle Pace, Susan Elliott, Zac Yates, Yi-Hong Han, Alain Balland, Wei Wang, Dean K. Pettit,

Viral Infectious Diseases and Gene Expression in Insects

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.