Affinity purification of novel bispecific antibodies recognising carcinoembryonic antigen and doxorubicin

We have developed a method which combines Protein A affinity chromatography and HPLC analytical and semipreparative hydroxyapatite affinity chromatogr aphy to purify bispecific antibodies (BsMabs) from hybrid-hybridomas secret ing antibodies recognising carcinoembryonic antigen (CEA) and the chemother apeutic drug doxorubicin (Dox). Elution of the HPLC hydroxyapatite columns with a 60-360 mM phosphate buffer gradient was found to give better separat ion than elution with a 60-180 mM phosphate buffer gradient. Careful monito ring of HPLC fractions by enzyme linked immunosorbent assays for anti-CEA, anti-Dox and dual anti-CEA/anti-Dox activity, and pooling of fractions on t he basis of these results, enabled the purification of novel BsMabs for use in in vitro and preclinical in vivo experiments. (C) 2001 Elsevier Science B.V. All rights reserved.