Assessment of chemical modifications of sites in the CDRs of recombinant antibodies: Susceptibility vs. functionality of critical quality attributes

• Published in: mAbs 2014-03, Vol.6 (2), p.327-339
• Main Authors: Haberger, Markus, Bomans, Katrin, Diepold, Katharina, Hook, Michaela, Gassner, Jana, Schlothauer, Tilman, Zwick, Adrian, Spick, Christian, Kepert, Jochen Felix, Hienz, Brigitte, Wiedmann, Michael, Beck, Hermann, Metzger, Philipp, Mølhøj, Michael, Knoblich, Constanze, Grauschopf, Ulla, Reusch, Dietmar, Bulau, Patrick
• Format: Article
• Language: English
• Subjects: Chromatography, Ion Exchange; Chromatography, Liquid; Complementarity Determining Regions - metabolism; critical quality attributes; deamidation; developability; glycation; Glycation End Products, Advanced - metabolism; Hot Temperature; Humans; Hydrogen-Ion Concentration; Immunoglobulin G - metabolism; Mass Spectrometry; oxidation; Oxidative Stress; Peptide Mapping - methods; protein degradation; Protein Processing, Post-Translational; Proteolysis; quality by design; recombinant antibodies; Recombinant Proteins - metabolism; Surface Plasmon Resonance
• ISSN: 1942-0862; 1942-0870; 1942-0870; 1942-0862
• DOI: 10.4161/mabs.27876

Modifications like asparagine deamidation, aspartate isomerization, methionine oxidation, and lysine glycation are typical degradations for recombinant antibodies. For the identification and functional evaluation of antibody critical quality attributes (CQAs) derived from chemical modifications in the complementary-determining regions (CDRs) and the conserved regions, an approach employing specific stress conditions, elevated temperatures, pH, oxidizing agents, and forced glycation with glucose incubation, was applied. The application of the specific stress conditions combined with ion exchange chromatography, proteolytic peptide mapping, quantitative liquid chromatography mass spectrometry, and functional evaluation by surface plasmon resonance analysis was adequate to identify and functionally assess chemical modification sites in the CDRs of a recombinant IgG1. LC-Met-4, LC-Asn-30/31, LC-Asn-92, HC-Met-100c, and HC Lys-33 were identified as potential CQAs. However, none of the assessed degradation products led to a complete loss of functionality if only one light or heavy chain of the native antibody was affected.