Production of Fab fragments of monoclonal antibodies using polyelectrolytecomplexes

A new method for the production of monovalent Fab fragments of antibodies h as been developed. Traditionally Fab fragments are produced by proteolytic digestion of antibodies in solution followed by isolation of Fab fragments. In the case of monoclonal antibodies against inactivated subunits of glyce raldehyde-3-phosphate dehydrogenase, digestion with papain resulted in sign ificant damage of the binding sites of the Fab fragments. Antigen was coval ently attached to the polycation, poly(N-ethyl-4-vinylpyridinium bromide). Proteolysis of monoclonal antibodies in the presence of the antigen-polycat ion conjugate followed by (i) precipitation induced by addition of polyanio n, poly(methacrylic) acid, and pH shift from 7.3 to 6.5 and (ii) elution at pH 3.0 resulted in 90% immunologically competent Fab fragments. Moreover, the papain concentration required for proteolysis was 10 times less in the case of antibodies bound to the antigen-polycation conjugate than that of f ree antibodies in solution. The digestion of antibodies bound to the antige n-polyelectrolyte complex was less damaging, suggesting that binding to the antigen-polycation conjugate not only protected binding sites of monoclona l antibodies from proteolytic damage but also facilitated the proteolysis p robably by exposing antibody molecules in a way convenient for proteolytic attack by papain. (C) 2000 Academic Press.