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Transient expression of human antibodies in mammalian cells

Nature Protocols volume 13, pages 99–117 (2018)Cite this article

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Abstract

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.

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Figure 1: Schematic overview of the procedure.
Figure 2: Antibody formats for expression in mammalian cells (overview).
Figure 3: Design of gene constructs.
Figure 4: Quality control of purified antibody preparations.
Figure 5: Optimization of heavy/light chain DNA transfection ratios (IgG format).

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Acknowledgements

This work was supported by the Australian Research Council and the National Health and Medical Research Council.

Author information

Authors and Affiliations

  1. Garvan Institute of Medical Research,, Sydney, Australia

    Rodrigo Vazquez-Lombardi, Damien Nevoltris, Ansha Luthra, Peter Schofield & Daniel Christ

  2. St Vincent' Clinical School, University of New South Wales, Sydney, Australia

    Ansha Luthra, Peter Schofield & Daniel Christ

  3. School of Business Administration, University of San Diego, San Diego, California, USA

    Carsten Zimmermann

  4. Rady School of Management, University of California San Diego, La Jolla, California, USA

    Carsten Zimmermann

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  1. Rodrigo Vazquez-Lombardi
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Contributions

R.V.-L., D.N., A.L., P.S. and C.Z. wrote the manuscript and generated figures. D.C. wrote the manuscript, generated figures and supervised the research.

Corresponding author

Correspondence to Daniel Christ.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Data 1

Sequences for construct design. (PDF 391 kb)

Supplementary Data 2

Herceptin IgG heavy chain.dna. (ZIP 8 kb)

Supplementary Data 3

Herceptin IgG light chain.dna. (ZIP 5 kb)

Supplementary Data 4

Herceptin Fab heavy chain.dna. (ZIP 5 kb)

Supplementary Data 5

Herceptin Fab light chain.dna. (ZIP 5 kb)

Supplementary Data 6

IL-2-Fc.dna. (ZIP 8 kb)

Supplementary Data 7

Fc-His.dna. (ZIP 7 kb)

Supplementary Data 8

Herceptin-Erbitux bispecific scFv.dna. (ZIP 12 kb)

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Vazquez-Lombardi, R., Nevoltris, D., Luthra, A. et al. Transient expression of human antibodies in mammalian cells. Nat Protoc 13, 99–117 (2018). https://doi.org/10.1038/nprot.2017.126

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