Development of Ion Mobility-Mass Spectrometry as a High Throughput Assay for Biotherapeutic Characterization

Throughout the development of therapeutic drugs, it is essential that their structures be rigorously characterized to ensure their safety and efficacy. Small molecule drugs are often straightforward to characterize due to their size, but drug materials produced through biological means, or biotherapeutics, are often orders of magnitude more complex making their analysis challenging. Biotherapeutics encompass a wide range of modalities, but monoclonal antibody (mAb) based structures have transformed the modern pharmaceutical industry. The benefits of mAbs in comparison to conventional small molecular therapies include their selectivity, efficacy, and limited toxicity. These characteristics have led to a host of mAb-based therapies that have enjoyed startling success for indications ranging from Alzheimer’s disease to cancer. The importance of such biotherapeutics have also highlighted the need to develop new technologies that can effectively assay the complex structures associated with mAbs and their generic alternatives, so-called biosimilars. Over the last twenty years mass spectrometry (MS) has emerged as a key tool for structural biology, enabling the rapid analysis of proteins and protein complexes. When coupled to ion mobility (IM), IM-MS is capable of measuring the shape, size, and charge of protein ions providing extraordinary details regarding protein conformation, binding, and post-translational status. By employing collision induced unfolding (CIU), a technique where protein ions undergo collisional heating in order to provoke conformational shifts in the ions studied, IM-MS becomes a technique capable of measuring protein stability - a key critical quality attribute for the development of biotherapeutics. However, the amount of information we can obtain and the speed in which we can perform these CIU experiments must be improved to meet the needs of biopharmaceutical discovery and development. In this thesis, we describe new enhancements to the throughput data processing, and information content for mAb therapeutics. In Chapter 2, we develop an empirical equation that predicts mAb ion charge stripping and enables the acquisition of CIU data in charge multiplexed mode, experiments which we pilot on a newly developed IM-MS platform. This approach provides a 120-fold improvement in the throughput of CIU data acquisitions. In Chapter 3, we develop the most comprehensive assessment yet attempted to describe the reproducibility of CIU data by accumulating over 1,000 total individual CIU datasets acquired for an innovator mAb and two associated biosimilars. Our work establishes that CIU can reproducibly differentiate biosimilars from innovator therapeutics when distinction is not possible using differential scanning calorimetry (DSC), and that CIU can follow the changes in mAb monomer structure caused upon heat stress. In chapter 4, we describe an IM-MS and CIU workflow for the biophysical characterization of biotherapeutic mAb dimer structures produced under a variety of stress conditions. Performing stress studies on two model IgG1 structures, we quantitate differences in their response to stress and in the levels of aggregate formed. Changes in CIU arrival time distribution widths and ground state collision cross sections (CCS) are stress dependent. Lastly, we compare our experimental measurements to computational models to unravel the structure of gas-phase mAb dimer structures. Overall, the development of the IM-MS and CIU methods described in this thesis will establish their utility in the future characterization of biotherapeutics.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/169711/1/dvallejo_1.pd