New Ion Mobility-Mass Spectrometry Tools to Characterize and Differentiate Similar Proteins

Abstract

This dissertation describes new ion mobility-mass spectrometry (IM-MS) approaches for the characterization and differentiation of similar proteins, namely antibodies. Antibodies form the basis of a growing class of biotherapeutics, and they pose an analytical challenge due to their size, flexibility, and heterogeneity. Thorough characterization is required for the safety and efficacy of such therapeutics. IM is a gas-phase separation technique in which charged ions are separated based on their size, shape, and charge, and from IM measurements, ions’ collision cross-sections, Ω, can be determined. When IM is paired with MS, which gives a readout of ions’ mass-to-charge (m/z) ratios, richer structural information can be gained. The new approaches described herein utilize various combinations of solution-phase chemistry, gas-phase chemistry, and collisional activation in conjunction with IM-MS to probe gas-phase ion structures and enhance the ability to distinguish between similar antibodies.Chapter 1 reviews the effects of charge on ion structures through the discussion of results from gas-phase charge-reduction experiments using cation-to-anion proton-transfer reactions (CAPTR). Comparisons with results from other charge-reduction strategies are also provided. Chapter 2 discusses the differentiation of IgG1κ and IgG4κ antibodies using combinations of CAPTR, collisional activation, and IM-MS. The novel application of a Jensen-Shannon similarity score for the quantitative comparison of apparent Ω distributions is also introduced. Pre-CAPTR and post-CAPTR activation are applied, and the quantitative comparison of Ω distributions reveals that, relative to established workflows using energy-dependent IM without charge-state manipulation, pre-CAPTR activation and post-CAPTR activation both enhance the differentiation of these antibodies by IM-MS. Chapters 3 and 4 discuss the use of reducing agents in native IM-MS for the maintenance of redox conditions in electrospray solutions and for the performance of disulfide bond reduction. Chapter 3 investigates the addition of three common reducing agents to protein electrospray ionization solutions and provides needed guidance on which reducing agents and concentrations minimize mass spectral signal interference and protein structural disruption. Chapter 4 introduces a novel programmed-temperature electrospray ionization (ptESI) source and a novel method for performing disulfide bond reduction of proteins with real-time monitoring by MS or IM-MS. The temperature and time resolution allows experimenters to view the onset and progression of bond reduction, rather than just the final product. Four antibody samples are subjected to real-time disulfide bond reduction, and the antibodies are easily distinguished by their reduction profiles, suggesting that this method is sensitive to the stability and disulfide-bond connectivity of these proteins. Overall, the new IM-MS methods presented here could be implemented to enhance current biotherapeutic characterization workflows.