Ion Mobility-Mass Spectrometry and Collision-Induced Unfolding of Designed Bispecific Antibody Therapeutics

Abstract

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.

Native ion mobility‐mass spectrometry reveals the binding mechanisms of anti‐amyloid therapeutic antibodies

“…By fitting sigmoid curves to the CIU data, we are able to extract CIU50 values which correspond to the accelerating potential necessary to convert 50% of the preceding CIU feature into the following state detected in the assay. Such values have been observed previously to report on domain‐specific stability values within the detected mAb:Aβ complexes (Villafuerte‐Vega et al, 2023 ). Analysis of this data reveals CIU50‐1 values of 63.9 ± 0.6 and 77.7 ± 1.2 as well as CIU50‐2 values of 97.7 ± 1.2 and 137.2 ± 3.2 for apo and 1:4 A34:Aβ 40 complexes respectively, indicating an increase in mAb stability upon binding (Figure 4d,e ) Such increases in stability are also observed for Adu and Cre‐Aβ complexes, although the extent of stabilization observed upon Aβ binding appears to be uniquely related to the mAb tested, with Adu and A34 increasing in stability to a greater extent than Cre upon Aβ 40 attachment when compared across identical complex stoichiometries (Figure 3f ).…”

“…In contrast, CIU50-2 values reflect the greater enhancement in mAb stability upon Aβ 40 binding for Adu and A34 referenced above (Figure 4b). Prior work has indicated that CIU50-1 and CIU50-2 values are related to Fab and Fc unfolding processes respectively, indicating non-local HOS involvement in Adu and A34 Aβ 40 binding (Villafuerte-Vega et al, 2023). The nIM-MS data described above has been focused on Aβ 40 , the most abundant Aβ proteoform present in vivo (Spies et al, 2010).…”

Investigation of Structure-Stabilizing Elements in Proteins by Ion Mobility Mass Spectrometry and Collision-Induced Unfolding

“…This proposed combined approach of CIU experiments and enzymatic digestion holds broad applicability to systems of pharmaceutical and biological significance. The workflow is also valuable in assessing biosimilars or protein analogues for specific applications. − …”

Fluorinated Ethylamines as Electrospray-Compatible Neutral pH Buffers for Native Mass Spectrometry

“…This study highlights the applicability of DFEA and TFEA as neutral pH buffers compatible with native ESI-MS. While many native ESI-MS experiments yield high-quality spectra over short acquisitions, some, like CIU experiments, often involve prolonged data acquisitions from a single loaded nano ESI tip. Without a suitable buffer, electrochemical reactions within the small volume loaded into the nano ESI tip might subject biomolecules to changing pH conditions for tens of minutes prior to being sprayed, potentially compromising the robustness of the measurement.…”