An integrated strategy for the process development of a recombinant antibody-cytokine fusion protein expressed in BHK cells
| Autor: | Glyn N. Stacey, A. V. Savage, M. Cuffe, C. Mielke, E. Rieke, J. B. Griffiths, T. Welge, C. Burger, D. Looby, José L. Moreira, Hansjörg Hauser, Manuel J.T. Carrondo, Elsa M. Dias, K. Hayes, Helder J. Cruz |
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| Rok vydání: | 1999 |
| Předmět: |
Glycosylation
Recombinant Fusion Proteins Genetic Vectors Molecular Sequence Data Oligosaccharides Antineoplastic Agents Biology Kidney Applied Microbiology and Biotechnology Culture Media Serum-Free Cell Line law.invention chemistry.chemical_compound Bioreactors law Cricetinae Cell Clone Gene expression Genes Synthetic Tumor Cells Cultured Baby hamster kidney cell Animals Secretion Selection Genetic Chromatography High Pressure Liquid Mesocricetus Tumor Necrosis Factor-alpha business.industry General Medicine Fusion protein Biotechnology ErbB Receptors Carbohydrate Sequence Genes Biochemistry chemistry Cell culture Immunoglobulin G Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Recombinant DNA Drug Screening Assays Antitumor business Protein Processing Post-Translational |
| Zdroj: | Applied Microbiology and Biotechnology. 52:345-353 |
| ISSN: | 1432-0614 0175-7598 |
| DOI: | 10.1007/s002530051530 |
| Popis: | Recombinant fusion proteins offer important new therapeutic approaches for the future. This report describes the use of three different genetic strategies (i.e. “mono-”, “bi-” and “tri-cistronic” vectors) to achieve stable secretion from BHK cells of a glycosylated antibody-cytokine fusion protein designed for use in anti-tumour therapy. It describes selection of a robust and effective production cell line based on stability of secretion of the product, quality of mRNA and protein products and performance in in vitro bioassays for potency. The data obtained at this stage were utilised in the selection of a suitable candidate production cell line. The relative productivity and general performance of the cells in stirred tank and fixed bed culture systems indicated that a variety of cell culture technologies provided robust tools for production of a highly selected cell clone. Consistency of the product glycosylation was determined by analysis of released oligosaccharides using matrix-assisted laser desorption ionisation – time of flight mass spectrometry and high-performance anion exchange chromatography. These investigations showed consistent expression of three glycoforms of the fusion protein which varied in their relative proportions in different culture systems and at different time points in a fixed bed reactor with continuous perfusion. In conclusion, this study dealt with a range of important scientific and technical issues which are essential for regulatory approval and commercial success of a recombinant protein and elucidates some useful markers for process development for similar recombinant biologicals. |
| Databáze: | OpenAIRE |
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