We have developed a method which combines Protein A affinity chromatography and HPLC analytical and semipreparative hydroxyapatite affinity chromatography to purify bispecific antibodies (BsMabs) from hybrid-hybridomas secreting antibodies recognising carcinoembryonic antigen (CEA) and the chemotherapeutic drug doxorubicin (Dox). Elution of the HPLC hydroxyapatite columns with a 60-360 mM phosphate buffer gradient was found to give better separation than elution with a 60-180 mM phosphate buffer gradient. Careful monitoring 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 the basis of these results, enabled the purification of novel BsMabs for use in in vitro and preclinical in vivo experiments.