Buffer-dependent fragmentation of a humanized full-length monoclonal antibody.
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 + 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.
Salinas
Branden A
BA
Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado, USA. branden.salinas@gmail.com
Sathish
Hasige A
HA
Shah
Ambarish U
AU
Carpenter
John F
JF
Randolph
Theodore W
TW
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D002021:Buffers; D002152:Calorimetry, Differential Scanning; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000737:chemistry
D000911:Antibodies, Monoclonal; D002021:Buffers; D007074:Immunoglobulin G; D014364:Tryptophan; D014508:Urea