Development of a Universal, CGMP-ready, clonal HEK293 Cell Line for the Manufacturing of Adeno-Associated Virus, Lenti- and Adenoviral Vectors

Utilizing the well-established, adherent, Frank Graham HEK293 cell line as a starting point, we have successfully adapted cells, from a qualified master cell bank, to grow in animal component-free, suspension conditions, in shake flasks and bioreactors. In headto- head studies comparing our SKPT-HEK suspension-adapted versus 3 commercially available suspension HEK293 cell lines, we demonstrate comparable yields of AAV vectors, following plasmid triple transfection method for virus production. In order to establish a cell line amenable for use in GMP-grade manufacturing, we clonally printed SKPT-HEK293 pool cells using the Cytena f.sight cell printing and CloneSelect imaging platform. Herein, we show full-traceability of clonal outgrowth from single-cell through scale-up and have identified select clones that perform equally well in head-to-head comparison studies versus other HEK293 cell lines for virus production. The lead clone 4G9, supports the robust production of all viral platform tested: AAV, LVV, and Adenovirus. Importantly, the 4G9 clone can support the production across a diverse panel of all natural AAV serotypes evaluated (AAV1, 2, 5, 6, 8, 9) as well as Lentiviral CAR-Ts. Establishment of a CGMP master cell banks for use in large scale AAV manufacturing is underway with expected availability in 2024. The advancement of this production-ready clone represents a significant cost saving for viral vector manufacturing. With integrated process and analytical development as well as commercial scale manufacturing, SK pharmteco can streamline the entire manufacturing process.