(157e) Advances and Opportunities in Adeno-Associated Virus (AAV) Manufacturing Process for Gene Therapy Application

Gene therapy has gained major traction from biopharmaceutical industries due to the encouraging prospect of curing numerous genetic disorders, potentially as a one-time treatment. Adeno-associated virus (AAV) is widely recognized as the most promising delivery vector for gene therapy to carry the therapeutic genetic payload as a package. A triple-transient transfection technique, with three different plasmids, employed on HEK293 cells is one of the most popular techniques for manufacturing AAV particles. However, several manufacturing challenges, such as poor packaging efficiency, low titer and poor yield of the full capsids of AAV, lead to higher cost, thereby limiting the accessibility of gene therapy to the global population.

At the Amgen Bioprocessing Center at Keck Graduate Institute (KGI), we focus on upstream and downstream process development for AAV to make gene therapy affordable. A triple-transient transfection technique was implemented with HEK293 cells to produce AAV Serotype-2 packaged with green fluorescent protein (GFP) gene as the gene of interest. After cell harvesting, lysing and clarification, AAV2 was obtained along with the dissolved process-related and product-related impurities. The impurities were removed by sequential affinity chromatography (AC) and anion exchange chromatography (AEXC) along with intermediate ultrafiltration/diafiltration (UF/DF) steps. The study demonstrated production and isolation of filled capsids of the viral vector. AAV titer in the range of 1x 10⁹-1x10¹⁰ vp/mL and 1x 10¹⁰-1x10¹¹ vp/mL for adherent HEK293 and suspension HEK293, respectively, were obtained from the cell culture study. A full to empty capsid ratio in the range of 15-20 % was observed. For downstream processing, a systematic approach evaluating the process parameters, such as type of elution buffer and gradient elution strategy, was implemented to design efficient chromatography steps. This presentation will primarily focus on upstream process development for AAV manufacturing. A design of experiment (DoE) study, with the help of JMP software, was conducted to evaluate the effect of different process parameters on AAV production. Effects of type of cell culture media, plasmid to host cell ratio, transfection reagent to host cell ratio and effect of addition of supplements, on AAV expression will be illustrated. Challenges associated with upstream and downstream processing of AAV, such as low titer, tendency to form aggregates, and difficulty in isolating the full capsids from the unwanted forms of the capsid will be discussed. Results obtained from various analytical techniques implemented for assessment of the quality attributes of AAV will be presented. Finally, scope of future studies for further advancement of viral vector manufacturing will be discussed.