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

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.

Author information

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.

Correspondence to Paul W H I Parren.

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All authors are Genmab employees and have stock and/or warrants in Genmab.

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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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