Transient expression of human antibodies in mammalian cells

• Published in: Nature protocols Vol. 13; no. 1; pp. 99 - 117
• Main Authors: Vazquez-Lombardi, Rodrigo, Nevoltris, Damien, Luthra, Ansha, Schofield, Peter, Zimmermann, Carsten, Christ, Daniel
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.01.2018; Nature Publishing Group
• Subjects: 42/109; 631/1647/664/2228; 631/250/2152/2153/1291; 631/61/185; 631/61/338/469; 631/61/51; 82/1; 82/16; 82/29; Affinity chromatography; Analytical Chemistry; Animal models; Animals; Antibodies; Antibodies - genetics; Antibodies - metabolism; Bacteria; Biological Techniques; Cell culture; Cells, Cultured; Chromatography, Affinity; Computational Biology/Bioinformatics; Contamination; Culture media; Electrophoresis, Polyacrylamide Gel - methods; Endotoxins; Gene expression; Genetic aspects; Genetic Vectors; Genotypes; Glycosylation; Health aspects; Humans; Immune response; Immunoglobulin Fab Fragments - genetics; Immunoglobulin Fab Fragments - isolation & purification; Immunoglobulin Fab Fragments - metabolism; Immunoglobulin G; Immunoglobulin G - genetics; Immunoglobulin G - metabolism; Interferometry; Interferometry - methods; Life Sciences; Mammalian cells; Mammals; Microarrays; Organic Chemistry; Phage display; Phages; Protein Engineering - methods; protocol; Purification; Reagents; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; System effectiveness
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2017.126

Mammalian cells are powerful expression systems for producing glycosylated recombinant antibody preparations with minimal endotoxin contamination. This protocol describes procedures for antibody design, expression, purification and characterization. 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 (>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.