Ion mobility-mass spectrometry and collision induced unfolding of designed bispecific antibody therapeutics

Bispecific antibodies (bsAbs) represent a critically important class of emerging therapeutics capable of simultaneously two different antigens simultaneously. As such, bsAbs have been developed as effective treatment agents for diseases that remain challenging for conventional monoclonal antibody (mAb) therapeutics to access. Despite this, bsAbs are intricate molecules, requiring both the appropriate engineering and pairing of linked heavy and light chains derived from separate parent mAbs. Current analytical tools for tracking the bsAb construction process have demonstrated a limited ability to robustly probe the higher order structure (HOS) of bsAbs. Native ion mobility-mass spectrometry (IM-MS) and collision induced unfolding (CIU) have proven to be useful tools in probing the HOS of mAb therapeutics. In this report, we de-scribe a series of comprehensive IM-MS and CIU datasets that reveal HOS details associated with knob-into-hole (KiH) bsAb model system. We find that quantitative analysis of CIU data indicates that global bsAb stability occupies and in-termediate space between the stabilities recorded for its parent mAbs. Furthermore, our CIU data identifies the hole-containing half of the bsAb construct to be least stable, thus driving much of the overall stability of the bsAb. An analysis of both intact bsAb and enzymatic fragments allows us to link the first and second CIU transitions observed for intact bsAbs to the Fab and Fc domains, respectively. This result is likely general for CIU data collected from low charge state mAb ions and is supported by data acquired for deglycosylated bsAb and mAb constructs, each of which of which indi-cate greater destabilization of the second CIU transition observed in our data. When integrated, our CIU analysis allows us to link changes in the first CIU transition primarily to the Fab region of the hole-containing halfmer, while the second CIU transition is likely strongly connected to the Fc region of the knob-containing half of the bsAb construct. Taken together, our results provide an unprecedented roadmap for evaluating the domain-level stabilities and HOS of both bsAbs and mAb constructs using CIU.