Fragmentation of a highly purified monoclonal antibody attributed to residual CHO cell protease activity |
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Authors: | Gao Sharon X Zhang Ying Stansberry-Perkins Kensey Buko Alex Bai Shujun Nguyen Vanessa Brader Mark L |
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Institution: | Department of Analytical Biochemistry, Biogen Idec, 5200 Research Place, San Diego, California, USA. sharon.gao@biogenidec.com |
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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. |
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Keywords: | monoclonal antibody fragmentation protease cleavage hinge region formulation |
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