Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2486374 | Journal of Pharmaceutical Sciences | 2009 | 13 Pages |
Abstract
We investigated the formation and stability of succinimide, an intermediate of deamidation events, in recombinant monoclonal antibodies (mAbs). During the course of an analytical development study of an IgG1 mAbs, we observed that a specific antibody population could be separated from the main product by cation-exchange (CEX) chromatography. The cell-based bioassay measured a â¼70% drop in potency for this fraction. Liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS) and tandem mass spectrometry (LC-MS/MS) analyses showed that the modified CEX fraction resulted from the formation of a succinimide intermediate at Asn 55 in the complementarity determining region (CDR) of the heavy chain. Biacore assay revealed a â¼50% decrease in ligand binding activity for the succinimide-containing Fab with respect to the native Fab. It was found that the succinimide form existed as a stable intermediate with a half-life of â¼3 h at 37°C and pH 7.6. Stress studies indicated that mildly acidic pH conditions (pH 5) favored succinimide accumulation, causing a gradual loss in potency. Hydrolysis of the succinimide resulted in a further drop in potency. The implications of the succinimide formation at Asn 55, a highly conserved residue among IgG1 (mAbs), are discussed. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3509-3521, 2009
Keywords
Related Topics
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Pharmacology, Toxicology and Pharmaceutical Science
Drug Discovery
Authors
Boxu Yan, Sean Steen, David Hambly, John Valliere-Douglass, Tim Vanden Bos, Scott Smallwood, Zac Yates, Thomas Arroll, Yihong Han, Himanshu Gadgil, Ramil F. Latypov, Alison Wallace, Aiching Lim, Gerd R. Kleemann, Weichun Wang, Alain Balland,