Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO-cell derived biotherapeutics
| Autor: | Qi Chen, Gregory S. Blank, Scott Lute, Bin Yang, Daniel Strauss, Douglas D. Frey, Cintia Ho, Kurt Brorson, Zinaida Tebaykina |
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| Rok vydání: | 2009 |
| Předmět: |
medicine.drug_class
viruses Virus Attachment Bioengineering CHO Cells Monoclonal antibody Applied Microbiology and Biotechnology Virus Sepharose Cricetulus Cricetinae medicine Animals Chromatography Chromatofocusing Chemistry Chinese hamster ovary cell Antibodies Monoclonal Chromatography Agarose Ligand (biochemistry) Culture Media Isoelectric point Biochemistry Cell culture Viruses Biotechnology |
| Zdroj: | Biotechnology and Bioengineering. 104:371-380 |
| ISSN: | 1097-0290 0006-3592 |
| DOI: | 10.1002/bit.22416 |
| Popis: | During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non-electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science-based process validation strategies to ensure viral safety of biotechnology products. |
| Databáze: | OpenAIRE |
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