How to Make Bispecific Antibodies

  • Ruth R. French
Part of the Springer Protocols Handbooks book series (SPH)


This protocol describes the production of bispecific F(ab’)2 antibody derivatives (BsAbs) by the linking of two Fab/tc fragments via their hinge region SH groups using the bifunctional crosslinker o-phenylenedimaleimide (o-PDM) as described by Glennie et al. (1,2). The procedure is illustrated in Fig. 1. The first step is to obtain F(ab’)2 from the two parent IgG antibodies. Methods for digestion of IgG to F(ab’)2 are described in  Chapter 151. Fab’ fragments are then prepared from the two F(ab’)2 species by reduction with thiol, thus exposing free SH groups at the hinge region (three SH-groups for mouse IgG1 and IgG2a antibodies) (see Note 1). One of the Faba species (Fab’-A) is selected for alkylation with o-PDM. Because o-PDM has a strong tendency to crosslink adjacent intramolecular SH-groups, two of the three hinge SH-groups will probably be linked together, leaving a single reactive maleimide group available for conjugation Fig. 1; see Note 2). Excess o-PDM is then removed by column chromatography, and the Fab’-A(mal) is mixed with the second reduced Fab’ (Fab’-B) under conditions favoring the crosslinking of the maleimide and SH groups. When equal amounts of the two parent Fab’ species are used, the major product is bispecific F(ab’)2, resulting from the reaction of one Fab’-A(mal) with one of the SH groups at the hinge of Fab’-B. Increasing the proportion of Fab’-A(mal) in the reaction mixture results in a significant amount of F(ab’)3 product by the reaction of two molecules of Fab’-A(mal) with two free SH-groups at the hinge of a single Fab’-B molecule (see Note 3). The remaining free SH groups on Fab’-B are alkylated, and the F(ab’)2 bispecific antibody product (Fab’-A×Fab’-B) is separated by gel filtration chromatography. Each stage of the procedure is checked by HPLC.


High Performance Liquid Chromatography High Performance Liquid Chromatography Hinge Region High Performance Liquid Chromatography Analysis Chart Recorder 
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  1. 1.
    Glennie, M. J., McBride, H. M., Worth, A. T., and Stevenson, G. T. (1987) Preparation and performance of bispecific F(ab’γ)2 antibody containing thioether-linked Fab’γ fragments. J. Immunol. 139, 2367–2375.PubMedGoogle Scholar
  2. 2.
    Glennie, M. J., Tutt, A. L., and Greenman, J. (1993) Preparation of multispecific F(ab’)2 and F(ab’)3 antibody derivatives, in Tumour Immunobiology, A Practical Approach (Gallagher, G., Rees, R. C., and Reynolds, C. W., eds.), Oxford University Press, Oxford, UK, pp. 225–244.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Ruth R. French
    • 1
  1. 1.Lymphoma Research UnitTenovus Research LaboratorySouthhampton General HospitalUK

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