Chemical production of bispecific antibodies.

This chapter discusses two related methods for creating Fab' x Fab' chemically linked BsAb. Both methods require the generation of purified F(ab')2 fragments of each antibody and use reagents that react with the free thiols generated upon reduction of interheavy chain disulfide bonds of the F(ab')2 fragments. Upon reduction, the resulting Fabs are then recombined to form a Fab' x Fab' BsAb. 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) acts to regenerate disulfide bonds between the two Fabs, whereas o-phenylenedimaleimide (o-PDM) acts to form a thioether bond between the two Fabs. After coupling, the bispecific antibody is purified from the uncoupled Fabs by size-exclusion chromatography. The advantages and disadvantages of each conjugation method are discussed.