Bispecific antibodies (bsAbs) represent a critically important class of emerging therapeutics capable of targeting 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 these advantages, bsAbs are intricate molecules, requiring both the appropriate engineering and pairing of 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 describe a series of detailed and quantitative IM-MS and CIU data sets that reveal HOS details associated with a knob-into-hole (KiH) bsAb model system and its corresponding parent mAbs. We find that quantitative analysis of CIU data indicates that global KiH bsAb stability occupies an intermediate space between the stabilities recorded for its parent mAbs. Furthermore, our CIU data identify the hole-containing half of the KiH bsAb construct to be the least stable, thus driving much of the overall stability of the KiH bsAb. An analysis of both intact bsAb and enzymatic fragments allows us to associate the first and second CIU transitions observed for the intact KiH bsAb to the unfolding Fab and Fc domains, respectively. This result is likely general for CIU data collected for low charge state mAb ions and is supported by data acquired for deglycosylated KiH bsAb and mAb constructs, each of which indicates 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 halfmer. Taken together, our results provide an unprecedented road map for evaluating the domain-level stabilities and HOS of both KiH bsAb and mAb constructs using CIU.

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.

Resistant starch is a prebiotic with breakdown by gut bacteria requiring the action of specialized amylases and starch-binding proteins. The human gut symbiont Ruminococcus bromii expresses granular starch-binding protein Sas6 (Starch Adherence System member 6) that consists of two starch-specific carbohydrate binding modules from family 26 (RbCBM26) and family 74 (RbCBM74). Here we present the crystal structures of Sas6 and RbCBM74 with a double helical dimer of maltodecaose bound along an extended surface groove. Binding data combined with native mass spectrometry suggest that RbCBM26 binds short maltooligosaccharides while RbCBM74 can bind single and double helical α-glucans. Our results support a model by which RbCBM74 and RbCBM26 bind neighboring α-glucan chains at the granule surface. CBM74s are conserved among starch granule-degrading bacteria and our work provides molecular insight into how this structure is accommodated by select gut species.