Advancing Mass Spectrometry Platforms for Next-Generation Biotherapeutics: Antibodies, AAVs, and RNA Therapeutics - Project Summary Biotherapeutics such as antibodies, RNA drugs, and adeno-associated viruses are transforming modern medicine, but their effectiveness depends on a fragile balance: biomolecular structure must be preserved through manufacturing, storage, and delivery. Even subtle structural changes can undermine therapeutic activity or trigger harmful immune responses. Current stability assays are limited because they capture global melting or activity loss but cannot directly connect failure pathways to molecular structure. This gap restricts our ability to understand why biotherapeutics fail and to design more resilient therapies. This project develops advanced mass spectrometry methods to reveal the molecular principles that govern stability in three major therapeutic classes: antibodies, messenger RNAs, and adeno-associated viruses (AAVs). The proposed platform integrates native mass spectrometry, ion mobility-mobility mass spectrometry, charge detection mass spectrometry, and controlled activation methods to track how biomolecules unfold, disassemble, or degrade under stress. By coupling gas-phase stability measurements to orthogonal solution-phase benchmarks such as differential scanning fluorimetry and circular dichroism spectroscopy, these approaches will connect structural descriptors directly to unfolding transitions, aggregation, and genome release events. For antibodies, we will map how sequence, glycosylation, and higher-order aggregation shape stability and link these features to changes in antigen binding. For RNAs, we will establish the first MS-based assays to quantify unfolding transitions of kilobase-scale transcripts and probe how encapsulation in lipid nanoparticles alters stability. For AAVs, we will define the molecular pathways of capsid destabilization and genome ejection, integrating experimental data with computational models to explain how different serotypes, genomes, and formulations confer resilience. We will also use the methods developed for RNA analysis to study AAV DNA genomes. This program is innovative because it unites emerging mass spectrometry technologies with established biophysical benchmarks to create a stability platform applicable across proteins, RNAs, and viral particles. By focusing on the shared molecular principles of resilience, the work will establish broadly applicable rules for predicting and improving biomolecular stability. These outcomes will strengthen the foundations of structural biology while providing new strategies for biopharmaceutical development and formulation.
