title: Fragmentation of a highly purified monoclonal antibody attributed to residual CHO cell protease activity.
abstract: Monoclonal antibody (mAb) fragmentation can be a widespread problem across the biotechnology industry and there is a current need to better understand the underlying principles. Here, we report an example of a high-purity human IgG1 mAb prepared from CHO cells exhibiting fragmentation that can be attributed to residual proteolytic enzyme activity. The concomitant occurrence of proteolytic and non-proteolytic peptide bond cleavage is shown and the respective fragmentation patterns characterized using high-resolution LC-MS. Fragmentation rates are monitored by SE-HPLC and SDS-PAGE over the pH range 4-6 and characterized in the presence and absence of pepstatin A, an inhibitor of acidic proteases. After 20 days at 40°C, pH 4, ∼60% decrease in BIIB-mAb monomer peak occurred attributed to residual proteolytic activity. At pH 5, this value was ∼13%. These results have implications for formulation design studies and the interpretation of accelerated stability data. A simple method to screen for acidic protease activity using the proteolytic enzyme inhibitor pepstatin A is described.
authors.affiliation: Department of Analytical Biochemistry, Biogen Idec, 5200 Research Place, San Diego, California, USA. sharon.gao@biogenidec.com
authors.forename: Sharon X
authors.initials: SX
authors.lastname: Gao
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Kensey
authors.initials: K
authors.lastname: Stansberry-Perkins
authors.forename: Alex
authors.initials: A
authors.lastname: Buko
authors.forename: Shujun
authors.initials: S
authors.lastname: Bai
authors.forename: Vanessa
authors.initials: V
authors.lastname: Nguyen
authors.forename: Mark L
authors.initials: ML
authors.lastname: Brader
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification / Q000378:metabolism*; D016466:CHO Cells / Q000201:enzymology*; D006224:Cricetinae; D003412:Cricetulus; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D010450:Endopeptidases