학술논문
Transient expression of human antibodies in mammalian cells
Document Type
Report
Author
Source
Nature Protocols. January 2018, Vol. 13 Issue 1, p99, 19 p.
Subject
Language
English
ISSN
1754-2189
Abstract
Author(s): Rodrigo Vazquez-Lombardi [1]; Damien Nevoltris [1]; Ansha Luthra [1, 2]; Peter Schofield [1, 2]; Carsten Zimmermann [3, 4]; Daniel Christ (corresponding author) [1, 2] Introduction The recombinant production of [...]
Recombinant expression of antibody molecules in mammalian cells offers important advantages over traditionally utilized bacterial expression, including glycosylation required for antibody functionality and markedly reduced levels of endotoxin contamination. Advances in transient mammalian expression systems enable high yields ( [greater than] 100 mg/liter) that now allow for effective recombinant antibody production at a reasonable cost. Here, we provide step-by-step protocols for the design and recombinant expression of full-length IgG antibodies and antibody-derived constructs (including Fab, Fc-fusions and bispecifics) in mammalian cells. Antibody constructs are designed by combining antibody variable domains, generated by phage display or derived from human/humanized monoclonals, with constant regions. The constructs are then expressed from mammalian vectors, secreted into culture media, purified by affinity chromatography and characterized by biolayer interferometry. This article provides detailed protocols, sequences and strategies that allow the expression and purification of endotoxin-free antibody reagents suitable for testing in animal models within a 3-week time frame. Keywords: Human antibody, Bispecific, Fc fusion protein, Fv, scFv, single-chain Fv, Variable fragment, Heavy chain, Light chain, Fragment antigen binding, Fab, Transient mammalian expression, Endotoxin, Protein A, Protein G, Protein L, IMAC, Immobilised metal ion affinity chromatography, Biolayer interferometry, antibody design, antibody purification, mammalian antibody, antibody production, recombinant antibody, IgG, endotoxin-free antibody
Recombinant expression of antibody molecules in mammalian cells offers important advantages over traditionally utilized bacterial expression, including glycosylation required for antibody functionality and markedly reduced levels of endotoxin contamination. Advances in transient mammalian expression systems enable high yields ( [greater than] 100 mg/liter) that now allow for effective recombinant antibody production at a reasonable cost. Here, we provide step-by-step protocols for the design and recombinant expression of full-length IgG antibodies and antibody-derived constructs (including Fab, Fc-fusions and bispecifics) in mammalian cells. Antibody constructs are designed by combining antibody variable domains, generated by phage display or derived from human/humanized monoclonals, with constant regions. The constructs are then expressed from mammalian vectors, secreted into culture media, purified by affinity chromatography and characterized by biolayer interferometry. This article provides detailed protocols, sequences and strategies that allow the expression and purification of endotoxin-free antibody reagents suitable for testing in animal models within a 3-week time frame. Keywords: Human antibody, Bispecific, Fc fusion protein, Fv, scFv, single-chain Fv, Variable fragment, Heavy chain, Light chain, Fragment antigen binding, Fab, Transient mammalian expression, Endotoxin, Protein A, Protein G, Protein L, IMAC, Immobilised metal ion affinity chromatography, Biolayer interferometry, antibody design, antibody purification, mammalian antibody, antibody production, recombinant antibody, IgG, endotoxin-free antibody