AAV Production – How To Improve Transfection Efficiency And Titer Yield

Traditionally, production of Adenoviral associated viral (AAV) vectors is achieved by triple transient transfection using the adherent HEK293 cell line in fetal bovine serum (FBS)-supplemented basal media, such as DMEM. The basal media provides nutritional support as well as buffering capacity for the cells while FBS provides the appropriate mitogenic and survival signaling. Collectively, this media/supplement combination supports HEK293 cell growth to achieve densities appropriate for transfection and subsequent vector production.

Although proven successful at small scale in terms of commercially viable AAV titers, use of this substance for large scale clinical manufacturing applications represents a significant safety risk due to potential adventitious agent contamination, unreliable supply chain from limited global supply, and considerable variability in vector yield due to lot-to-lot composition variations. Though FBS composition has considerable variability, several key protein components have been thoroughly characterized and the functional contribution in cell culture media is well understood.

Here we demonstrate how poorly defined and blood-derived components can be replaced for chemically defined and blood free solutions in adherent HEK-based AAV production.