Developing Structural Mass Spectrometry Approaches for Therapeutic Antibody Characterization

Abstract

Therapeutic monoclonal antibodies (mAbs) and mAb-based therapeutics represent the fastest growing class of biopharmaceuticals over the past few decades. In comparison with the small molecule drugs, these large complex biomolecules offer higher specificity, efficacy, and fewer side effects, owing to their diverse compositions and modes of action. The comprehensive structural characterization of therapeutic antibodies is of critical importance for the successful discovery and development of such biopharmaceuticals, yet poses many challenges to modern measurement science. To address these challenges, in this dissertation we develop structural mass spectrometry-based approaches, including ion mobility-mass spectrometry (IM-MS) combined with collision induced unfolding (CIU) methods, and hydrogen/deuterium exchange mass spectrometry (HDX -MS), for assessing the structure, stability, and dynamics of such therapeutics. First, we demonstrated the capacity of CIU for characterizing intact antibodies with minimal sample preparation and purification. In Chapter 2, four human IgG isoforms containing different disulfide bridges were rapidly differentiated using CIU. The CIU analysis was also able to capture the conformational changes in the mAb induced by the removal of entire glycans from the protein. Furthermore, we validated this CIU protocol through a systematic statistical evaluation of its reproducibility. In Chapter 3, we evaluated the ability of CIU to detect the impact of subtle changes to N-linked glycan composition on overall antibody structure. Subtle structural alterations and decrease in gas-phase stabilities were detected in both intact mAbs and antibody Fc fragments, as a result of sequential eliminations of terminal sugars in the glycosylation. We found evidence for a strong correlation between the gas-phase stabilities of antibody ions and the amount of sugars attached to their sequences. We continued by developing native IM-MS methods in combination with CIU analysis for characterization antibody drug conjugates (ADCs) in Chapter 4. By using mAb-biotin conjugates as model ADCs, we show that despite nearly identical ground state structures, subtle structural differences can be detected upon biotin conjugation using CIU. Our analysis involves the global comparison of CIU datasets, width analysis of the arrival time distributions, and the detection of gas-phase unfolding transitions. We then integrated the native IM-MS and CIU approaches with other state-of-the-art MS techniques for a comprehensive evaluation of the biosimilar and originator infliximab in Chapter 5. These two mAb products displayed identical primary structures, highly similar modifications, comparable higher order structures, and similar degradation pathways under stress. However, the differences in the glycoforms and Fc receptor bindings were observed, suggesting potential differences in the efficacy profiles for these two drugs. Finally, a novel HDX-MS strategy was developed in Chapter 6 for high concentration mAb formulations. This method was used to study the liquid-liquid phase separation behavior of an IgG4. Decreased deuterium uptake levels were observed for mAbs in the high-density phase compared to those in the depleted lower density phase, revealing evidence of conformational changes for this mAb as a function of overall protein concentration.