Bioprocess Strategies to Mitigate the Impact of Proteases on Adeno-Associated Viral (AAV) Vector VP1 Capsid Content

A handful of studies have suggested that cellular and baculovirus proteases may impact the capsid nature of insect cell-produced AAV gene therapy viral vectors. The aim of this investigation was to detect the presence of extracellular protease activity in cell culture, and design bioprocess strategies that may inhibit protease activity, and by extension prevent the reduction of AAV VP1 capsid protein which has been determined to be important for virus infectivity. 


We attempted late cell culture process changes (i.e., temperature shifts and pH shifts) to modulate protease activity. We also tried addition of inorganic salts that may have a similar effect. During AAV production, the addition of the cysteine proteinase inhibitor E64 to cell culture led to an increase of normalized AAV VP1 levels by 50-70% compared to the control. A similar outcome was evidenced when the cell culture was exposed to a 23°C shift. Time-course analysis of VP1 levels showed that E64 and temperature shift prevented the continuous reduction of VP1 levels, which was seen in the control condition. Moreover, the late addition of an inorganic salt at micromolar concentration had a similar effect on VP1 content. We confirmed that the salt addition also reduced extracellular protease activity within the last days of the process. Similar trends were seen during the production of a second AAV serotype