Buffer-dependent fragmentation of a humanized full-length monoclonal antibody
Corresponding Author
Branden A. Salinas
Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado
Center for Pharmaceutical Biotechnology, University of Colorado, Boulder, Colorado
Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado. Telephone: 303-492-7471; Fax: 303-492-4341.Search for more papers by this authorHasige A. Sathish
Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado
MedImmune, Inc., Gaithersburg, Maryland 20878
Center for Pharmaceutical Biotechnology, University of Colorado, Boulder, Colorado
Search for more papers by this authorJohn F. Carpenter
Center for Pharmaceutical Biotechnology, University of Colorado, Boulder, Colorado
University of Colorado Health Sciences Center, Denver, Colorado
Search for more papers by this authorTheodore W. Randolph
Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado
Center for Pharmaceutical Biotechnology, University of Colorado, Boulder, Colorado
Search for more papers by this authorCorresponding Author
Branden A. Salinas
Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado
Center for Pharmaceutical Biotechnology, University of Colorado, Boulder, Colorado
Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado. Telephone: 303-492-7471; Fax: 303-492-4341.Search for more papers by this authorHasige A. Sathish
Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado
MedImmune, Inc., Gaithersburg, Maryland 20878
Center for Pharmaceutical Biotechnology, University of Colorado, Boulder, Colorado
Search for more papers by this authorJohn F. Carpenter
Center for Pharmaceutical Biotechnology, University of Colorado, Boulder, Colorado
University of Colorado Health Sciences Center, Denver, Colorado
Search for more papers by this authorTheodore W. Randolph
Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado
Center for Pharmaceutical Biotechnology, University of Colorado, Boulder, Colorado
Search for more papers by this authorAbstract
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. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:2962–2974, 2010
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