Fig. 3

Biolayer interferometry (BLI) analysis of trastuzumab and trastuzumab-Fc variants to evaluate hFcRn binding kinetics. a Initial association rates (on-rates) of trastuzumab, trastuzumab-DHS, trastuzumab-PFc29, trastuzumab-YML, and trastuzumab-EML were determined at pH 6.0 using the Octet R8 system. hFcRn-His was immobilized onto Ni-NTA biosensors, and Fc variants diluted in PBS (pH 6.0) were introduced for 20 s. Early association rates were calculated based on the first 2 s of the sensorgram. b Initial dissociation rates (off-rates) of trastuzumab and Fc variants were measured at pH 7.4. After the association phase, biosensors were transferred to PBS (pH 7.4) for 5 s, and early dissociation rates were calculated based on the first 1 s of the dissociation phase. Trastuzumab-YML exhibited the fastest on-rate and off-rate among all Fc variants, reflecting its enhanced pH-selective binding kinetics. Error bars represent the standard deviations calculated from quadruplicate experiments