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Fragmentation of a highly purified monoclonal antibody attributed to residual CHO cell protease activity
Author(s) -
Gao Sharon X.,
Zhang Ying,
StansberryPerkins Kensey,
Buko Alex,
Bai Shujun,
Nguyen Vanessa,
Brader Mark L.
Publication year - 2011
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22982
Subject(s) - pepstatin , fragmentation (computing) , proteases , chemistry , protease , monoclonal antibody , biochemistry , proteolytic enzymes , enzyme , microbiology and biotechnology , proteolysis , chromatography , biology , antibody , immunology , ecology
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. Biotechnol. Bioeng. 2011; 108:977–982. © 2010 Wiley Periodicals, Inc.