(T1030-08-43) An Efficient HEK293 Cell Line for Scalable, GMP-Ready Production of Adeno-Associated Viral Vectors

Purpose: Adeno-associated virus (AAV) vector-based gene therapy has shown great promise in a multitude of clinical trials and approved therapeutics. Many developments, including novel plasmid vector systems and AAV serotypes, as well as scalable vector production methods, have continually advanced the field to not only increase the purity and potency of recombinant AAV vectors, but also produce AAV in high quantities. Along with these ongoing developments, additional effort has been focused on creating efficient cell lines that can be readily transfected for high-yielding and improved quality vector production. The goal of the project was to develop an efficient clone of HEK293 cells that can support scalable, GMP-ready viral vector production.

Methods: A fully characterized master cell bank was made from an in-house subclone chosen for higher productivity vg/ml (ddPCR) and % full compared to other subclones made from the same parental clone. For clonal comparison, in-house model viral vectors were produced via triple plasmid transfection in suspension culture at the 2L scale using the highly productive clone, other selected top clones, and a commercially available cell line. To confirm scalability, the top clone was scaled up to a 200L bioreactor for vector production using a platform process. The downstream process steps included tangential flow filtration, affinity chromatography (AAVX) and polishing chromatography (CIM Q4). Vector particle titer outcomes measured from day 3 total cell lysates were used to estimate % full, along with post-purification analytical ultracentrifugation (AUC) measurements. Additional analytical tests for comparisons included residual impurities such as host cell DNA (hcDNA).

Results: Selection of the highly productive subclone was made based on (i) the vector genome titer, (ii) higher % full, (iii) clone scalability, and (iv) hcDNA content. Our results showed that with AAV5 vectors, the in-house subclone achieved 2X productivity, combined with 2X % full capsids, and a 10X reduction in hcDNA compared to the commercially available cell line using a relatively unoptimized system.

Conclusion: Using a highly productive clone for AAV production can improve efficiency of the AAV manufacturing process and the critical quality attributes of the viral vector. When combined with a standardized platform process for viral vector manufacturing, an efficient HEK293 cell line can help expedite the first-to-clinic path for AAV vector-based gene therapies.

Acknowledgements:Acknowledge collaboration with Madison site of Catalent.