Fragmentation of a highly purified monoclonal antibody attributed to residual CHO cell protease activity
| Autor: | Kensey Stansberry-Perkins, Alex Buko, Ying Zhang, Mark L. Brader, Vanessa Nguyen, Shujun Bai, Sharon X. Gao |
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| Rok vydání: | 2010 |
| Předmět: |
Proteases
medicine.medical_treatment Bioengineering CHO Cells Cleavage (embryo) Applied Microbiology and Biotechnology chemistry.chemical_compound Cricetulus Cricetinae Endopeptidases medicine Peptide bond Animals Humans chemistry.chemical_classification Protease biology Chemistry Proteolytic enzymes Antibodies Monoclonal Enzyme Biochemistry Enzyme inhibitor Immunoglobulin G biology.protein Pepstatin Biotechnology |
| Zdroj: | Biotechnology and bioengineering. 108(4) |
| ISSN: | 1097-0290 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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