Measuring Size and Shape Heterogeneity in AAVs with Ion Mobility Mass Spectrometry

Adeno-associated viruses (AAVs) are at the forefront of biopharmaceutical development as gene therapy vectors. The successful approval of these medicines requires robust characterization including an assessment of therapeutic gene content. Using ion mobility–mass spectrometry (IM-MS) techniques, we determine empty:full capsid ratios and explore structural differences in AAV particles relevant to payload. With drift tube IM-MS, we demonstrate that empty capsids, while slightly smaller, present more conformational variability than full capsids. This method also resolves two partially filled capsid species with intermediate masses, between that of empty and full capsids wherein the lower mass feature is conformationally more like empty and the higher mass feature appears highly similar to the full capsid. We present data using travelling wave ion mobility (TWIM) operated under conditions that limit or favour the mass-to-charge ratio (m/z) dependence on ion transit time through the drift cell via the velocity relaxation effect. This permits separation of empty and full capsids in the absence of mass spectrometry information, providing a new approach to study conformational and mass differences in super massive particles.