Identification and monitoring of host cell proteins by mass spectrometry combined with high performance immunochemistry testing
| Autor: | Patrick Bulau, Kyrillos Kyriosoglou, Katharina Diepold, Dietmar Reusch, Lisa Adolf, Gabriele Eberl, Harald Wegele, Christian Schmalz, Katrin Bomans, Veronika Roedl, Antje Lang, Ulrike Strauss, Rudolf Vogel, Michael Molhoj, Michael Wiedmann |
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| Rok vydání: | 2013 |
| Předmět: |
Phosphatase
lcsh:Medicine Enzyme-Linked Immunosorbent Assay Biology medicine.disease_cause Chromatography Affinity Mass Spectrometry Liquid chromatography–mass spectrometry Immunochemistry medicine Escherichia coli lcsh:Science chemistry.chemical_classification Multidisciplinary Escherichia coli Proteins lcsh:R Biological activity Alkaline Phosphatase Yeast Biological Therapy Enzyme chemistry Biochemistry Alkaline phosphatase lcsh:Q Research Article |
| Zdroj: | PLoS ONE PLoS ONE, Vol 8, Iss 11, p e81639 (2013) |
| ISSN: | 1932-6203 |
| Popis: | Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP) was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day) manner. |
| Databáze: | OpenAIRE |
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