Molecular Insights into the Thermal Stability of mAbs with Variable‐Temperature Ion‐Mobility Mass Spectrometry

Abstract

The aggregation of protein-based therapeutics such as monoclonal antibodies (mAbs) can affect the efficacy of the treatment and can even induce effects that are adverse to the patient. Protein engineering is used to shift the mAb away from an aggregation-prone state by increasing the thermodynamic stability of the native fold, which might in turn alter conformational flexibility. We have probed the thermal stability of three types of intact IgG molecules and two Fc-hinge fragments by using variable-temperature ion-mobility mass spectrometry (VT-IM-MS). We observed changes in the conformations of isolated proteins as a function of temperature (300-550 K). The observed differences in thermal stability between IgG subclasses can be rationalized in terms of changes to higher-order structural organization mitigated by the hinge region. VT-IM-MS provides insights into mAbs structural thermodynamics and is presented as a promising tool for thermal-stability studies for proteins of therapeutic interest.

Mass spectrometry locates local and allosteric conformational changes that occur on cofactor binding

“…Gaussian curves were fitted to the most intense CCSDs for the apo form and retained across other charge states and cofactor stoichiometries to highlight the distinguishable conformations with reference to the apo form. This procedure has been used previously for protein complexes 15 and monomeric proteins 16 17 . The smallest conformer, shown by the purple curve centred on 4,550 Å 2 , contributes to the [2M+20H] 20+ and [2M+21H] 21+ charge states; it contributes more to [2M+21H] 21+ Fdc1 UbiX than Fdc1, suggesting that this small conformation is stabilized in this charge state by the cofactor.…”

Selectivity over coverage in de novo sequencing of IgGs

“…The in-source trapping desolvation voltage displays an optimum in terms of ECD at À100 V, likely a consequence of small structural changes and cleavage enhancement occurring as the effective temperature of the ion is raised. 33 The ions were then transmitted into the ECD cell where they were subjected to electron capture dissociation. The small permanent magnets ensure the radial connement of electrons emitted by a cathode while additional electrodes ensure longitudinal connement and efficient ion transfer to the HCD cell.…”

“…We hypothesized that a possible explanation could involve differences in the conformations and structural dynamics of the IgG molecules. 33,41,54,55 In the absence of extensive collisional activation, low energy (<1 eV) electron capture dissociation is known to result in the preferential release of fragments from the surface-exposed regions of native proteins. 17,[45][46][47] The rationale is that exposed domains and chains have fewer non-covalent interactions upon transition to the gas phase, and therefore more readily release backbone fragments upon electron attachment.…”

Adding a new separation dimension to MS and LC–MS: What is the utility of ion mobility spectrometry?

“…Variable temperature IMS (VT‐IM–MS) were also reported to address mAb conformation and conformational transitions induced upon IM cell temperature variations . In this work, the thermal stability of three types of intact IgG molecules (with the same antigen‐binding specificity) has been probed by variable‐temperature IM–MS.…”