SEMIPREPARATIVE PURIFICATION AND VALIDATION OF MONOCLONAL-ANTIBODIES FOR IMMUNOTHERAPY IN MICE

A number of rat hybridomas were adapted to grow in RPMI containing eit her 5% IgG-depleted FCS or 1% serum-free Nutridoma. Alternatively, pro tein-free Ultradoma PF was used. Growth in these media allowed purific ation procedures to be used that are based on tangential ultrafiltrati on in combination with affinity chromatography on gels linked to prote in G or anti-rat L chain coupled antibodies. The isolated antibody pre parations were found to be pure and to consist of monomeric intact IgG . The yield and recovery of mAb using this procedure were found to be consistently high. These antibody preparations were analyzed for endot oxin contamination. Whereas during isolation endotoxin contamination i ncreased, the endotoxin content per mg purified protein did not. Affin ity chromatography on Detoxi-gel resulted in the efficient removal of this contamination and using this protocol the antibody preparations o btained were found to be of sufficient purity, activity and low endoto xin content to permit their in vivo use in animal models of immunother apy.