Buffer-dependent fragmentation of a humanized full-length monoclonal antibody

✍ Scribed by Branden A. Salinas; Hasige A. Sathish; Ambarish U. Shah; John F. Carpenter; Theodore W. Randolph

• Book ID: 102399645
• Publisher: John Wiley and Sons
• Year: 2010
• Tongue: English
• Weight: 298 KB
• Volume: 99
• Category: Article
• ISSN: 0022-3549
• DOI: 10.1002/jps.22056

✦ Synopsis

During storage stability studies of a monoclonal antibody (mAb) it was determined that the primary route of degradation involved fragmentation into lower molecular weight species. The fragmentation was characterized with size-exclusion high performance liquid chromatography (SE-HPLC), SDS-PAGE, and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. Fragmentation proceeded via hydrolysis, likely catalyzed by trace metal ions, of a peptide bond in the hinge region of the mAb's heavy chain, which produced two prominent low molecular weight species during storage: a single, free Fab fragment and a Fab þ Fc fragment. The fragmentation is observed in phosphate-buffered solutions at two ionic strengths but not in histidine-buffered solutions at identical ionic strengths. Chaotrope-induced and thermally induced unfolding studies of the mAb indicated differences in the unfolding pathways between the two buffer solutions. The folding intermediate observed during chaotrope-induced unfolding was further characterized by intrinsic fluorescence quenching, which suggested that a small portion of the molecule is resistant to chaotrope-induced unfolding in histidine buffer systems. The thermally induced unfolding indicates a reduction in cooperativity of the unfolding process in the presence of histidine relative to phosphate. A relationship between the histidine-induced effects on unfolding pathway and the relative resistance to fragmentation is suggested.