Solid phase synthesis of bifunctional antibodies

Abstract

Bifunctional antibodies were prepared using the principle of solid-phase synthesis. The two Fab′ fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab′) 2 fragments from intact IgG were prepared using an immobilized pepsin column. Goat, mouse and human antibodies were digested completely within 4 h. The F(ab′) 2 fragments thus produced did not contain any IgG impurities. The Fab′ fragments were produced by reducing the inter-heavy chain disulfide bonds using 2-mercaptoethylamine. The use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of the bifunctional antibody preparation and the rapid optimization of reaction conditions.