title: Buffer-dependent fragmentation of a humanized full-length monoclonal antibody.
abstract: 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.
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado, USA. branden.salinas@gmail.com
authors.forename: Branden A
authors.initials: BA
authors.lastname: Salinas
authors.forename: Hasige A
authors.initials: HA
authors.lastname: Sathish
authors.forename: Ambarish U
authors.initials: AU
authors.lastname: Shah
authors.forename: John F
authors.initials: JF
authors.lastname: Carpenter
authors.forename: Theodore W
authors.initials: TW
authors.lastname: Randolph
mesh_terms: 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
chemical_list: D000911:Antibodies, Monoclonal; D002021:Buffers; D007074:Immunoglobulin G; D014364:Tryptophan; D014508:Urea