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Updated: Aug 23, 2026

Laboratory Scale Production and Purification of a Therapeutic Antibody
Published on: January 24, 2017
Chemical production of bispecific antibodies
Robert F Graziano1, Paul Guptill
1Medarex Inc., Bloomburg, NJ, USA.
Abstract:
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.
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