Detailed Analytical Characterization of a Bispecific IgG1 CrossMab Antibody of the Knob-into-Hole Format Applying Various Stress Conditions Revealed Pronounced Stability

[Image: see text] In recent years, a variety of new antibody formats have been developed. One of these formats allows the binding of one type of antibody to two different epitopes. This can for example be achieved by introduction of the “knob-into-hole” format and a combined CrossMab approach. Due to their complexity, these bispecific antibodies are expected to result in an enhanced variety of different degradation products. Reports on the stability of these molecules are still largely lacking. To address this, a panel of stress conditions, including elevated temperature, pH, oxidizing agents, and forced glycation via glucose incubation, to identify and functionally evaluate critical quality attributes in the complementary-determining and conserved regions of a bispecific antibody was applied in this study. The exertion of various stress conditions combined with an assessment by size exclusion chromatography, ion exchange chromatography, LC–MS/MS peptide mapping, and functional evaluation by cell-based assays was adequate to identify chemical modification sites and assess the stability and integrity, as well as the functionality of a bispecific antibody. Stress conditions induced size variants and post-translational modifications, such as isomerization, deamidation, and oxidation, albeit to a modest extent. Of note, all the observed stress conditions largely maintained functionality. In summary, this study revealed the pronounced stability of IgG1 “knob-into-hole” bispecific CrossMab antibodies compared to already marketed antibody products.