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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.

Collision cross-sections (CCS) of immunoglobulins G1 and G4 have been determined using linear drift-tube ion-mobility mass spectrometry. Intact antibodies and Fc-hinge fragments present with a larger range of CCS than proteins of comparable size. This is rationalized with MD simulations, which indicate significant in vacuo dynamics between linked folded domains. The IgG4 subclass presents over a wider CCS range than the IgG1 subclass.

RATIONALE Non-covalent mass spectrometry (MS) offers considerable potential for protein-ligand screening in drug discovery programmes. However, there are some limitations with the time-of-flight (TOF) instrumentation typically employed that restrict the application of non-covalent MS in industrial laboratories. METHODS An Exactive Plus EMR mass spectrometer was investigated for its ability to characterise non-covalent protein–small molecule interactions. Nano-electrospray ionisation (nanoESI) infusion was achieved with a TriVersa NanoMate. The transport multipole and ion lens voltages, dissociation energies and pressure in the Orbitrap™ were optimised. Native MS was performed, with ligand titrations to judge retention of protein-ligand interactions, serial dilutions of native proteins as an indication of sensitivity, and a heterogeneous protein analysed for spectral resolution. RESULTS Interactions between native proteins and ligands are preserved during analysis on the Exactive Plus EMR, with the binding affinities determined in good agreement with expected values. High spectral resolution allows baseline separation of adduct ions, which should improve the accuracy and limit of detection for measuring ligand interactions. Data are also presented showing baseline resolution of glycoforms of a highly glycosylated protein, allowing binding of a fragment molecule to be detected. CONCLUSIONS The high sensitivity and spectral resolution achievable with the Orbitrap technology confer significant advantages over TOF mass spectrometers, and offer a solution to current limitations regarding throughput, data analysis and sample requirements. A further benefit of improved spectral resolution is the possibility of using heterogeneous protein samples such as glycoproteins for fragment screening. This would significantly expand the scope of applicability of non-covalent MS in the pharmaceutical and other industries. Copyright © 2014 John Wiley & Sons, Ltd.

The initial stages of drug discovery are increasingly reliant on development and improvement of analytical methods to investigate protein–protein and protein–ligand interactions. For over 20 years, mass spectrometry (MS) has been recognized as providing a fast, sensitive and high-throughput methodology for analysis of weak non-covalent complexes. Careful control of electrospray ionization conditions has enabled investigation of the structure, stability and interactions of proteins and peptides in a solvent free environment. This critical review covers the use of mass spectrometry for kinetic, dynamic and structural studies of proteins and protein complexes. We discuss how conjunction of mass spectrometry with related techniques and methodologies such as ion mobility, hydrogen–deuterium exchange (HDX), protein footprinting or chemical cross-linking can provide us with structural information useful for drug development. Along with other biophysical techniques, such as NMR or X-ray crystallography, mass spectrometry provides a powerful toolbox for investigation of biological problems of medical relevance (204 references).