Monoclonal antibodies (mAbs) are among the fastest growing class of therapeutics due to their high specificity and low incidence of side effects. Unlike most drugs, mAbs are complex macromolecules (∼150 kDa), leading to a host of quality control and characterization challenges inherent in their development. Recently, we introduced a new approach for the analysis of the intact proteins based on ion mobility-mass spectrometry (IM-MS). Our protocol involves the collision induced unfolding (CIU) of intact antibodies, where collisional heating in the gas-phase is used to generate unfolded antibody forms, which are subsequently separated by IM and then analyzed by MS. Collisional energy is added to the antibody ions in a stepwise fashion, and "fingerprint plots" are created that track the amount of unfolding undergone as a function of the energy imparted to the ions prior to IM separation. In this report, we have used these fingerprints to rapidly distinguish between antibody isoforms, possessing different numbers and/or patterns of disulfide bonding and general levels of glycosylation. In addition, we validate our CIU protocols through control experiments and systematic statistical evaluations of CIU reproducibility. We conclude by projecting the impact of our approach for antibody-related drug discovery and development applications.

Ion mobility-mass spectrometry (IM-MS) is a technology of growing importance for structural biology, providing complementary 3D structure information for biomolecules within samples that are difficult to analyze using conventional analytical tools through the near-simultaneous acquisition of ion collision cross sections (CCSs) and masses. Despite recent advances in IM-MS instrumentation, the resolution of closely related protein conformations remains challenging. Collision induced unfolding (CIU) has been demonstrated as a useful tool for resolving isocrossectional protein ions, as they often follow distinct unfolding pathways when subjected to collisional heating in the gas phase. CIU has been used for a variety of applications, from differentiating binding modes of activation state-selective kinase inhibitors to characterizing the domain structure of multidomain proteins. With the growing utilization of CIU as a tool for structural biology, significant challenges have emerged in data analysis and interpretation, specifically the normalization and comparison of CIU data sets. Here, we present CIUSuite, a suite of software modules designed for the rapid processing, analysis, comparison, and classification of CIU data. We demonstrate these tools as part of a series of workflows for applications in comparative structural biology, biotherapeutic analysis, and high throughput screening of kinase inhibitors. These examples illustrate both the potential for CIU in general protein analysis as well as a demonstration of best practices in the interpretation of CIU data.

In April 2016, the Food and Drug Administration approved the first biosimilar monoclonal antibody (mAb) – Inflectra/Remsima (Celltrion) based off the original product Remicade (infliximab, Janssen). Biosimilars promise significant cost savings for patients, but the unavoidable differences between innovator and copycat biologics raise questions regarding product interchangeability. In this study, Remicade and Remsima were examined by native mass spectrometry, ion mobility and quantitative peptide mapping. The levels of oxidation, deamidation and mutation of individual amino acids were remarkably similar. We found different levels of C-terminal truncation, soluble protein aggregates and glycation that all likely have a limited clinical impact. Importantly, we identified over 25 glycoforms for each product and observed glycoform population differences, with afucosylated glycans accounting for 19.7% of Remicade and 13,2% of Remsima glycoforms, which translated into a 2-fold reduction in FcγRIIIa binding for Remsima. While this difference was acknowledged in Remsima regulatory filings, our glycoform analysis and receptor binding results appear to be somewhat different from the published values, likely due to methodological differences between laboratories and improved glycoform identification by our laboratory using a peptide map-based method. Our mass spectrometry based analysis provides rapid and robust analytical information vital for biosimilar development. We have demonstrated the utility of our multiple attribute monitoring workflow using the model mAbs Remicade and Remsima, and have provided a template for analysis of future mAb biosimilars.

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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. Mass spectrometry has evolved into a rapid and sensitive tool for assessing the structures, stabilities, and dynamics of such proteins. Here, we review the current state-of-the-art mass spectrometry technologies focusing on the characterization of antibody-based therapeutics. We conclude by discussing the future of structural mass spectrometry, and its role in enabling the biopharmaceutical pipeline.