Method Qualification Of Mass Photometry For The Determination Of Full-To-Empty Capsid Ratio For rAAVs Using The Refeyn System

By Christine Zach, Sebastian Ritter, Tobias Klötzler, Alexandra Schoberth, and Sonya M. Schermann

The heterogeneity in packaging during the production of recombinant adeno-associated viruses (rAAV) can result in capsids containing DNA fragments of varying lengths. Methods for calculating full-to-empty ratios typically classify capsids into two categories: full capsids, which contain DNA close to the size of the full-length vector, and empty capsids, which contain little to no DNA. Consistent full-to-empty ratios are critical for ensuring the safety and efficacy of the final drug product, making reliable methods for monitoring this quality attribute essential.

Mass photometry (MP), based on the principles of interference reflection microscopy and interferometric scattering microscopy, offers a straightforward, rapid platform for analyzing full-to-empty ratios in rAAV samples with minimal sample input. MP measures the mass of individual biomolecular particles in their native state without the need for labels, making it ideal for assessing the quality of rAAV production. We validated this method according to ICHQ2 (R1) guidelines, using rAAV samples in typical production matrices and buffers.