Controlled Fab-arm exchange for the generation of stable bispecific IgG1
| Autor: | Ewald T. J. van den Bremer, Rob N. de Jong, Janine Schuurman, Arnout F. Gerritsen, Paul W. H. I. Parren, Joyce I. Meesters, Aran F. Labrijn, Muriel D. van Kampen, Patrick Priem |
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| Rok vydání: | 2014 |
| Předmět: |
Quality Control
Bispecific antibody Chemistry Genetic Vectors Disulfide bond Oxidation reduction Protein Engineering Combinatorial chemistry Molecular biology General Biochemistry Genetics and Molecular Biology Mass Spectrometry Immunoglobulin Fab Fragments Bioreactors HEK293 Cells Immunoglobulin G Bench scale Antibodies Bispecific Humans Point Mutation Oxidation-Reduction |
| Zdroj: | Nature protocols. 9(10) |
| ISSN: | 1750-2799 |
| Popis: | Labrijn et al. describe the generation of bispecific antibodies through controlled Fab-arm exchange, involving separate expression of two parental IgG1s containing single matching point mutations, followed by recombination of the 'half molecules'. The generation of bispecific antibodies (bsAbs) with natural IgG architecture in a practical and efficient manner has been a longstanding challenge. Here we describe controlled Fab-arm exchange (cFAE), which is an easy-to-use method to generate bispecific IgG1 (bsIgG1). The protocol involves the following: (i) separate expression of two parental IgG1s containing single matching point mutations in the CH3 domain; (ii) mixing of parental IgG1s under permissive redox conditions in vitro to enable recombination of half-molecules; (iii) removal of the reductant to allow reoxidation of interchain disulfide bonds; and (iv) analysis of exchange efficiency and final product using chromatography-based or mass spectrometry (MS)–based methods. The protocol generates bsAbs with regular IgG architecture, characteristics and quality attributes both at bench scale (micrograms to milligrams) and at a mini-bioreactor scale (milligrams to grams) that is designed to model large-scale manufacturing (kilograms). Starting from good-quality purified proteins, exchange efficiencies of ≥95% can routinely be obtained within 2–3 d (including quality control). |
| Databáze: | OpenAIRE |
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