Next-Generation Native Ion Mobility-Mass Spectrometry Methods for the Characterization of Biotherapeutics

Biopharmaceuticals, including therapies based on proteins and nucleic acids, are now among the most important classes of modern medicines. While they are used to treat a wide range of diseases, they are most commonly employed in cancer, autoimmune disorders, and infectious diseases, generating over $100 billion in annual sales. Despite these successes, many challenges loom in the discovery of next-generation biotherapeutics. We lack routine technologies that can quickly assess the sequences, chemical modification states, and higher- order structures (HOSs) of protein and nucleic acid products. Some products are known to produce a wide range of non-covalent complexes with targets, further complicating the refinement of their potency and the evaluation of their potential safety. Most biopharmaceuticals target membrane proteins, which are generally refractive to standard structural biology tools and may alter their structures in a manner dependent upon local lipid environment changes, which can thus influence characterization efforts, safety screening, and ultimate efficacy. Nucleic acid drugs (NADs) specifically are often packaged within lipid nanoparticles (LNPs), which have largely unknown influences on NAD structures and stabilities. This application seeks to close the above-noted gaps in technology through the development of new methods based on native ion mobility-mass spectrometry (nIM-MS), a technology platform that can quickly separate and measure both the sizes and the masses of complex biotherapeutic samples. Our approaches will also rely upon collision induced unfolding (CIU), which can be used to quickly assess the structures and stabilities of proteins and nucleic acids. Specifically, we plan to 1) develop rapid, droplet-based enzymatic chemistries for nIM-MS enabled structural analysis of monoclonal antibody (mAb) therapeutics, 2) produce improved access to mAb sequence and chemical modification information by leveraging rapid liquid chromatography and top-down MS approaches based on cyclic IM-MS, and 3) evaluating both the role of lipids on antibody-antigen complex formation and NAD HOS, through the creation of nIM-MS approaches compatible with lipid nanodiscs, vesicles, and nanoparticles. Our efforts will result in a battery of new nIM-MS methods that enable the complete and rapid evaluation of biotherapeutic sequence, modification status, binding preferences, and stabilities, thus enabling a new chapter in the development of biopharmaceuticals for the treatment of human disease. Project Number: 1R35GM163797-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Brandon Ruotolo | Institution: UNIVERSITY OF MICHIGAN AT ANN ARBOR, ANN ARBOR, MI | Award Amount: $565,993 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MCST-Q (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11330048