Enhanced Stability Differentiation of Therapeutic Polyclonal Antibodies with All Ion Unfolding-Ion Mobility-Mass Spectrometry.

Abstract

Compared with monoclonal antibodies, polyclonal antibodies (pAbs) have rather significant characteristics, including lower cost, shorter production cycle, and higher affinity. Therefore, to facilitate their applications in clinic, it is equally critical to comprehensively characterize the conformational stabilities of pAb at the molecular weight-resolved scale, which is technically challenging due to the lack of an effective analytical tool capable of simultaneously providing both stability and molecular weight information within an acceptable error range. Ion mobility-mass spectrometry (IM-MS) has grown as an alternative to rapidly assess protein conformational stability with accurate molecular weight information maintained, especially when equipped with a collision-induced unfolding (CIU) regime. Dynamic and transient conformational intermediates can be captured with the CIU-IM-MS technique, adding to traditional static structural measurements with collisional cross section. Most CIU-IM-MS-centered protocols are focusing on the application to isolated, targeted protein ions, namely, analyzing one single charge state at one time, limiting its analytical throughput and speed. In this study, we employed an enhanced unfolding regime, all ion unfolding (AIU), capable of the simultaneous operation of numerous ions at a time during stepped unfolding processes to analyze pAb. Results show that AIU can quantitatively characterize the subtle differences in conformational stability among four structurally similar pAbs with improved resolving capability by around a 2-4-fold increment in both stability and structure differentiating parameters. Besides, AIU also benefits from considerably saved time cost and improved spectrum quality with an elevated signal-to-noise ratio.