Chemical Production of Bispecific Antibodies

This chapter discusses two related methods for creating Fab′ � 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′ � 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.