Adeno-associated virus serotype 9 structural heterogeneity and stability characterized by charge detection mass spectrometry

The variability in structure and stability of adeno-associated virus serotype 9 (AAV9) in response to changes in buffer composition, pH, and temperature was investigated using charge detection mass spectrometry (CDMS). AAV9 virus-like particles (VLPs) consisting of only viral protein (VP) 3 and wild-type AAV9 capsids (i.e., capsids containing varying stoichiometries of VP1, VP2, and VP3) showed differences in structure, indicating that these different VP stoichiometries and compositions may contribute substantially to conformational heterogeneity. Significant differences in AAV9 structure and stability were observed in ammonium acetate (AA) vs. phosphate buffered saline (PBS) solutions under some conditions. At 37°C under acidic conditions, AAV capsids fell apart in AA, whereas in PBS, capsids underwent structural compaction. Subsequent nuclease binding experiments indicated that partially extruded DNA was the likely origin of this structural compaction that occurred under different physical and chemical conditions. Results from one freeze-thaw cycle indicated that the capsids degraded by a similar mechanism to that in acidified solution. The structural complexity revealed by CDMS highlights the advantages of this biophysical characterization method in providing, for the first time, a holistic insight into the potential heterogeneous conformational transitions of AAV9 during purification, storage, and the natural infection process.