Development of novel bispecific antibody (bsAb) platforms offers unprecedented opportunities for a wide variety of therapeutic applications. However, the expression and manufacturing of bsAbs with desired structures can be challenging. Owing to the uniqueness of each bsAb platform, more comprehensive and customized structural characterization is particularly important to understand the chemical or biological reactivity of bsAbs, as well as to guide process development, risk assessment, and manufacturing. In this work, we performed higher order structure characterization of the Regeneron bsAb platform with Fc site-specific substitutions through hydrogen deuterium exchange mass spectrometry (HDX-MS). Structural deprotection was identified at the CH2-CH3 interface in the Fc domain, owing to the site-specific substitutions. The structural deprotection was found to correlate with the decreased conformational stability of Fc domain. Under oxidative and thermal stress conditions, the Met residues located near the structurally deprotected region were identified to be susceptible to oxidation. In addition, the introduction of substitutions in the bsAb Fc resulted in a slight reduction of its binding affinity to the neonatal Fc receptor (FcRn). The detailed structural elucidation by HDX-MS improves understanding of the structure-property relationship of the Regeneron bsAb format, thus greatly aiding in process development, risk assessment, and manufacturing.
Keywords: Bispecific antibody; Fc conformation; FcRn binding; HDX-MS; methionine oxidation; site-specific substitution; thermal stability.