Structural and Functional Characterization of a Hole–Hole Homodimer Variant in a “Knob-Into-Hole” Bispecific Antibody

Bispecific antibodies have great potential to be the next-generation biotherapeutics due to their ability to simultaneously recognize two different targets. Compared to conventional monoclonal antibodies, knob-into-hole bispecific antibodies face unique challenges in production and characterization due to the increase in variant possibilities, such as homodimerization in covalent and noncovalent forms. In this study, a storage- and pH-sensitive hydrophobic interaction chromatography (HIC) profile change was observed for the hole–hole homodimer, and the multiple HIC peaks were explored and shown to be conformational isomers. We combined traditional analytical methods with hydrogen/deuterium exchange mass spectrometry (HDX MS), native mass spectrometry, and negative-staining electron microscopy to comprehensively characterize the hole–hole homodimer. HDX MS revealed conformational changes at the resolution of a few amino acids overlapping the CH2-CH3 domain interface. Conformational heterogeneity was also a...