Complementary methods for monitoring hole-hole homodimer associated with a WuXiBody-based asymmetric bispecific antibody

WuXiBody is a bispecific antibody (bsAb) platform developed by WuXi Biologics. Its key feature is the replacement of one parental antibody's CH1/CL region with the T-cell receptor (TCR) constant domain, which prevents mispairing between non-cognate heavy chain and light chain. In addition, heavy chain heterodimerization in asymmetric WuXiBody molecule is promoted by the knobs-into-holes (KiH) technology. Despite the great success of KiH strategy in improving heterodimer formation, homodimers (especially the hole-hole homodimer) can still be generated at low levels. In general, detection and monitoring of homodimers during KiH bsAb purification are challenging as homodimers share similar physicochemical properties with the target heterodimeric bsAb. Nevertheless, the unique design of WuXiBody allows homodimers to be effectively detected and monitored by multiple methods. In the current work, with an asymmetric WuXiBody case study, we demonstrated that hole-hole homodimer can be effectively monitored by six chromatography methods including hydrophobic interaction chromatography (HIC), reverse phase (RP), cation exchange (CEX), KappaSelect, CaptureSelect CH1-XL and Protein L.