Improving Aggregation Control of Recombinant Adeno‐Associated Virus Serotype 2 (rAAV2) With Small Sugars and Ionic Salts

ABSTRACT Two persistent challenges in adeno‐associated virus (AAV) manufacturing are AAV particle aggregation and the separation of full and empty AAV capsids in ion‐exchange (IEX) chromatography processes, which add to AAV purification, formulation, and quality control challenges. AAV empty capsids and AAV aggregates are both considered product‐related impurities by regulatory agencies. AAV full capsids, which contain the genetic payload, is the AAV species that has the therapeutic value. Thus, it is necessary to continuously improve the control of AAV aggregation and the ratio of full to empty capsids during AAV downstream bioprocessing. We investigated a novel approach that significantly improves aggregation control of AAV serotype 2 (AAV2). The novel approach consisted of a systematic study, involving Design of Experiment (DoE), using common formulation excipients (namely, small sugars and ionic salts) to understand the effect of critical process parameters (excipient type, excipient concentration, and pH) on a critical quality attribute (AAV aggregates). With this approach, we observed a statistically significant reduction in AAV2 particle aggregation in solution. Results suggest that this aggregation‐control approach could provide insight into potentially being able to create a new strategy for improving the separation of full and empty AAV2 capsids in anion exchange (AEX) chromatography.