(T1230-07-42) Characterizing Potency of Model AAVs Using Imaging Flow Cytometry

Purpose: Adeno-associated viral vector (AAV) gene therapies encompass a diverse range of serotypes and transgenes. The current paradigm for gene therapy products is a heavy reliance on ultra-cold (-70°C) storage placing a burden on the supply chain and ability to reach patients. This burden can be reduced as we identify more advantageous formulations that will allow for higher temperature storage and longer shelf lives. To identify better formulations, a way to characterize drug product stability is needed, and potency is the most important critical quality attribute. Measuring potency allows for the quantification of the biological activity of the drug product providing a distinct advantage over alternative approaches.

Methods: An imaging flow cytometer was used to characterize potency of model AAVs of various serotypes containing a ZsGreen transgene. To do this, HEK293 cells were first plated and allowed to grow until confluent, then AAVs were added, and the ZsGreen containing transgene is infected into the HEK293 cells. After 48 hours the cells were harvested and imaged on a fluorescence microscope as a qualitative check prior to being run on the imaging flow cytometer. Flow cytometry was able to provide the percentage of cells out of the population that were ZsGreen+.

Results: This procedure was applied to model AAV2 stored at 40ºC for 1 and 3 weeks. When compared to AAV2 at time zero a sharp drop in the percentage of cells expressing ZsGreen was seen after 1 week at 40ºC and a further drop was seen after 3 weeks.

Conclusion: These data show how imaging flow cytometry enables the quantification of the drop in potency of AAV2 at 40ºC.This adds a tool to our toolbelt that enables the evaluation of various formulations on various serotypes of AAVs to identify a more robust drug product formulation.

Figure 2 lays out the infectivity procedure to prepare AAV infected cells for characterization through Flow Cytometry. HEK293 cells are grown to >80% confluence and then plated onto 6-well plates. Once the wells reach 90% confluence AAV is added. The amount added is based off the cell count and the desired MOI. 24-48 hours post AAV addition the cells are harvested and analyzed via flow cytometry.



Figure 1 contains different analytical techniques that can be used to characterize AAVs. These assays each characterize different critical quality attributes that can be used to determine AAV stability.



Flow cytometry was used to characterize AAV2 placed on stability at 40C for 3 weeks and AAV2 stored at 80C for 1 hour. Images that were taken on a fluorescent microscope and laid over brightfield images of the same cells are used to qualitatively compare the flow cytometry results. Prior to stability 70% of cells were zsGreen+. After 1 week at 40C that number dropped to 4.1% and after 3 weeks it is 0.5%. Showing a drop in potency when AAV2 is stored at 40C