Assessing AAV stability with mass photometry: Striking insights into the outcomes of heated capsids

The stability of adeno-associated virus (AAV) preparations is an essential attribute that requires thorough investigation. Typically, AAV stability is probed by heating the AAV sample and monitoring its genomic release and/or capsid disintegration. In recent years, mass photometry has emerged as a valuable tool to study various properties of AAV preparations – offering accurate results in minutes and using very little sample. In this webinar, we explore using mass photometry to assess capsid stability after heating different empty and filled AAV preparations. 

Quantifying empty and genome-filled AAV particles following heat treatment can be difficult due to the lack of a heat-resistant internal standard. We applied a heat-stable DNA plasmid as a reference during sample heating and mass photometry measurements. Following heating and AAV acquisition on the SamuxMP, we quantitatively determined the impact of thermal stress on AAVs. 

Empty AAVs demonstrated higher thermal stability at elevated temperatures than genome-filled capsids. For some AAV preparations, the decline of genome-filled capsids was accompanied by the detection of the released genome. At the same time, our data indicated that, upon AAV genome release, the capsids did not necessarily transform into empty AAVs but seemed to aggregate or disintegrate. Strikingly, some heated AAVs were disrupted capsids with the genome still bound, a feature that only emerged experimentally following incubation with a nuclease.

Our data demonstrate that the thermal uncoating process is highly AAV specific (i.e., can be influenced by serotype, genome, host system), and that mass photometry, which works for all serotypes, can be helpful in developing (custom) biochemistry assays (e.g., thermal stability) and delineating AAV properties. We argue that nuclease treatment in combination with mass photometry can be used as an additional analytical tool for assessing the structural integrity of recombinant and/or clinical AAV vectors.