Development Of A Universal, CGMP-Ready, Clonal HEK293 Cell Line For The Manufacturing Of Adeno-Associated Virus, Lenti- And Adenoviral Vectors

By Marguerite Campbell, Kelly Geosits, Ngan Trinh, Khan Umaer, Srivanya Tummala, Thomas J. Burton, Kaitlyn Krewson, Ryann Russell, and Brian Tomkowicz

Starting with the well-known adherent Frank Graham HEK293 cell line, we adapted cells from a qualified master cell bank to grow in animal component-free suspension conditions, suitable for shake flasks and bioreactors. Comparative studies of our SKPT-HEK suspension-adapted line with three commercial HEK293 suspension lines showed similar AAV vector yields using the plasmid triple transfection method.

To develop a CGMP-compliant cell line, we used Cytena’s f.sight cell printing and CloneSelect imaging to clonally isolate SKPT-HEK293 cells. We established traceable clonal outgrowth, identifying clones that matched other HEK293 lines in virus production, with the lead clone, 4G9, demonstrating robust production across AAV, LVV, and Adenovirus platforms. The 4G9 clone supports a wide range of natural AAV serotypes (AAV1, 2, 5, 6, 8, 9) and CAR-T Lenti-viral vectors. A CGMP master cell bank for large-scale AAV manufacturing is expected in 2024, offering a cost-effective solution for viral vector production with integrated process and analytical development from SK pharmteco.