Ion Mobility-Mass Spectrometry and Collision-Induced Unfolding Rapidly Characterize the Structural Polydispersity and Stability of an Fc-Fusion Protein.

Fc-fusion proteins are an emerging class of protein therapeutics that combine the properties of biological ligands with the unique properties of the fragment crystallizable (Fc) domain of an immunoglobulin G (IgG). Due to their diverse higher-order structures (HOSs), Fc-fusion proteins remain challenging characterization targets within biopharmaceutical pipelines. While high-resolution biophysical tools are available for HOS characterization, they frequently demand extended time frames and substantial quantities of purified samples, rendering them impractical for swiftly screening candidate molecules. Herein, we describe the development of ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) workflows that aim to fill this technology gap, where we focus on probing the HOS of a model Fc-Interleukin-10 (Fc-IL-10) fusion protein engineered using flexible glycine-serine linkers. We evaluate the ability of these techniques to probe the flexibility of Fc-IL-10 in the absence of bulk solvent relative to other proteins of similar size, as well as localize structural changes of low charge state Fc-IL-10 ions to specific Fc and IL-10 unfolding events during CIU. We subsequently apply these tools to probe the local effects of glycine-serine linkers on the HOS and stability of IL-10 homodimer, which is the biologically active form of IL-10. Our data reveals that Fc-IL-10 produces significantly more structural transitions during CIU and broader IM profiles when compared to a wide range of model proteins, indicative of its exceptional structural dynamism. Furthermore, we use a combination of enzymatic approaches to annotate these intricate CIU data and localize specific transitions to the unfolding of domains within Fc-IL-10. Finally, we detect a strong positive, quadratic relationship between average linker mass and fusion protein stability, suggesting a cooperative influence between glycine-serine linkers and overall fusion protein stability. This is the first reported study on the use of IM-MS and CIU to characterize HOS of Fc-fusion proteins, illustrating the practical applicability of this approach.

The Ruminococcus bromii amylosome protein Sas6 binds single and double helical α-glucan structures in starch.

Resistant starch is a prebiotic accessed by gut bacteria with specialized amylases and starch-binding proteins. The human gut symbiont Ruminococcus bromii expresses Sas6 (Starch Adherence System member 6), which 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 of RbCBM74 bound with a double helical dimer of maltodecaose. The RbCBM74 starch-binding groove complements the double helical α-glucan geometry of amylopectin, suggesting that this module selects this feature in starch granules. Isothermal titration calorimetry and native mass spectrometry demonstrate that RbCBM74 recognizes longer single and double helical α-glucans, while RbCBM26 binds short maltooligosaccharides. Bioinformatic analysis supports the conservation of the amylopectin-targeting platform in CBM74s from resistant-starch degrading bacteria. Our results suggest that RbCBM74 and RbCBM26 within Sas6 recognize discrete aspects of the starch granule, providing molecular insight into how this structure is accommodated by gut bacteria.

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 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.