Impact of electrostatics on the aggregation, genome release, and self-interactions of AAV9 capsids

Understanding solution conditions that enable favorable biophysical characteristics is important for formulation development of biologics and is a relatively new area for adeno-associated virus (AAV) products compared to more traditional therapeutic proteins. Solution pH and ionic strength have a considerable impact on electrostatic interactions and thus capsid-capsid interactions, which can potentially influence aggregation propensity. We have examined the impact of electrostatic interactions on the aggregation, genome release, and self-interaction behavior of AAV9 capsids. Aggregation at pH 4.5 after temperature stress was potentially exacerbated by a large population of empty/partial capsids, while at pH 7.4 degradation via genome loss was more likely. Additionally, decreasing the ionic strength from 150 mM to 5 mM caused reversible aggregate formation, though pH significantly impacted the salt concentration required for aggregate reversal. Static light scattering revealed capsid-capsid self-interactions are driven by strong electrostatic attractions at 150 mM for both pH 4.5 and 7.4, with stronger capsid-capsid interactions at pH 4.5. We employed a coarse-grained molecular model which predicted that capsid self-interactions are more sensitive to the change of ionic strength in the low/medium regime at pH 4.5 compared to pH 7.4. Overall, our data supports that electrostatic interactions play a key role in the stability of AAV9 capsids.