Author/Authors :
Yan، نويسنده , , Boxu and Valliere-Douglass، نويسنده , , John and Brady، نويسنده , , Lowell H. Steen، نويسنده , , Sean and Han، نويسنده , , Mei and Pace، نويسنده , , Danielle and Elliott، نويسنده , , Susan and Yates، نويسنده , , Zac and Han، نويسنده , , Yihong and Balland، نويسنده , , Alain and Wang، نويسنده , , Weichun and Pettit، نويسنده , , Dean، نويسنده ,
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.
Keywords :
peptide mapping , glycosylation , LC-TOF MS , Antibody , RP-HPLC , deamidation , Oxidation , cyclization , Isomerization
