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With adeno-associated viruses (AAVs) gaining ground as vehicles for gene therapy, it is increasingly critical to establish ways of controlling AAV quality and purity to ensure their safety and efficacy. Mass analysis could be an important first step in resolving the molecular architecture and transgene cargo of recombinant AAVs. However, such analysis is challenged by mass heterogeneity arising from variability in capsid composition and gene packaging, post-translational modifications, or residual co-purified impurities.

This webcast will demonstrate how the mass of AAV capsids can be determined using two different novel, emerging single-particle techniques: charge-detection mass spectrometry (CDMS) and mass photometry. In contrast to ensemble-based approaches (e.g., native mass spectrometry), mass photometry and CDMS can measure the mass of numerous, individual AAV particles, circumventing issues driven by sample heterogeneity.

Detection of AAVs’ charge and mass-over-charge ratio was done on an Orbitrap mass analyzer (the Q Exactive™ UHMR), commonly used for macromolecular analysis, while mass photometry, based on the microscopic detection of particles’ induced light scattering, was done on a recently developed AAV-dedicated mass photometer (the SamuxMP).

Three preparations of AAVs of two different serotypes (AAV8 and AAV2) were assessed in a side-by-side manner to directly compare measurement techniques and AAV production systems. Together, the data confirm that the different AAV production methods result in diverse AAV particle distributions, especially for filled AAVs. While founded on distinct physical principles, CDMS and mass photometry measurements prove to be in striking agreement.

• How different AAV production systems (e.g., insect versus mammalian) yield substantial differences in capsid composition and gene packing efficiency
• How the generation of recombinant AAVs with a transgene can lead to substantial under or overfilling
• How single-particle techniques, such as Orbitrap-based CDMS and mass photometry can determine AAV mass and capsid filling