PREFERENTIAL SPECIES-RESTRICTED HEAVY LIGHT-CHAIN PAIRING IN RAT MOUSE QUADROMAS - IMPLICATIONS FOR A SINGLE-STEP PURIFICATION OF BISPECIFIC ANTIBODIES

Conventional mouse/mouse or rat/rat hybrid-hybridoma supernatants cont ain up to 10 different IgG molecules consisting of various combination s of heavy and light chains. Hence, the yield of functional bispecific Ab is low, and purification is often complicated, hampering a general preclinical evaluation of, e.g., bispecific Ab-mediated tumor immunot herapy in animal models. In experiments to overcome this drawback we f ound that fusion of rat with mouse hybridomas opens the possibility of large scale production of bispecific Ab due to the increased incidenc e of correctly paired Ab and facilitated purification. In essence, rat /mouse quadroma-derived bispecific Ab have the following advantages: 1 ) enrichment of functional bispecific Ab because of preferential speci es-restricted heavy/light chain pairing (observed in four of four rat- mouse quadromas) in contrast to the random pairing in conventional mou se/mouse or rat/rat quadromas, and 2) a possible one-step purification of the quadroma supernatant with protein A. This simple chromatograph y step does not bind unwanted variants with parental rat/rat heavy cha in configuration, and the desired rat/mouse bispecific Ab are retained , which can then easily be separated from parental mouse Ab by sequent ial pH elution.