A Robust And Scalable Research Grade Platform Process For AAV6 Production And Purification

By Shona Robinson, James Fasano, Matthew Guilleman, Lisa Santry, Amira Rghei, Cherylene A. Plewa, Daniel Kennedy, Theresa Dao, Jay Cai, Azam Hassaninasab, Bojiao Yin, Mercedes Segura, Stacie L Seidel

Adeno-associated virus (AAV) is the most used vector in the field of gene therapy, with applications to treat and cure monogenetic disorders. There are currently hundreds of clinical trials underway for recombinant AAV (rAAV) therapeutics, in addition to a several clinically (EMA and FDA) approved products. Despite these clinical advancements in the gene therapy space, manufacturing of these vectors continues to be an ever-evolving challenge. Moreover, there is a clear need for robust, scalable, and cost-effective production of research grade material for indicative proof-of-concept work that is highly representative of process development platform material.

Many variables influence the success of rAAV vector production, including, but not limited to, production cell line, cell density, plasmid systems, including plasmid ratios and total plasmid DNA, media composition, and plasmid sequence composition. In the downstream process additional variables can include harvest and lysis conditions, filtration, stabilization and storage, purification method, and post-purification processing.

Here we describe research grade and process development productions of rAAV6, both based on transient transfection of serum-free cells grown in suspension. We demonstrate the progression of our well-established methods, including modifications to transfection process and purification method. Using a rAAV packaging genome sizes of 4.2kb- 4.7 kb, containing representative therapeutics , we have been able to consistently achieve titers of >7x1010 vector genomes (vg)/mL from crude production, with on average 55% and 65% yield after affinity batch purification in our research grade platform referred to as ElevateBio (EB) Batch, and AKTA capture chromatography in our process development (PD) platform, respectively.