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Controlled Fab-arm exchange for the generation of stable bispecific IgG1

Nature Protocols volume 9, pages 2450–2463 (2014)Cite this article

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Abstract

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).

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Figure 1: Generation of bsAbs by cFAE.
Figure 2: Workflow for the generation of bsAbs by cFAE.
Figure 3: Representation of the mini-bioreactor system.
Figure 4: cFAE in a mini-bioreactor system.
Figure 5: Characterization of bsIgG1 batches.
Figure 6: Quantification of 2-MEA.

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Acknowledgements

We thank M. Schuller for expert technical assistance and R. Hibbert, W. Vos, P. v. Berkel and M. Gramer for expert advice and helpful discussions.

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Authors and Affiliations

  1. Genmab, Utrecht, the Netherlands

    Aran F Labrijn, Joyce I Meesters, Patrick Priem, Rob N de Jong, Ewald T J van den Bremer, Muriel D van Kampen, Arnout F Gerritsen, Janine Schuurman & Paul W H I Parren

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  1. Aran F Labrijn
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Contributions

A.F.L., R.N.d.J., E.T.J.v.d.B., M.D.v.K., A.F.G., J.S. and P.W.H.I.P. designed the research; J.I.M., P.P., E.T.J.v.d.B. and M.D.v.K. carried out the experiments; A.F.L., J.I.M., P.P., R.N.d.J., E.T.J.v.d.B., M.D.v.K., A.F.G., J.S. and P.W.H.I.P. interpreted the data; A.F.L., J.I.M., P.P. and E.T.J.v.d.B. wrote the manuscript.

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Correspondence to Paul W H I Parren.

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Labrijn, A., Meesters, J., Priem, P. et al. Controlled Fab-arm exchange for the generation of stable bispecific IgG1. Nat Protoc 9, 2450–2463 (2014). https://doi.org/10.1038/nprot.2014.169

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