(97e) Downstream Purification Process Development for Adeno-Associated Virus (AAV)

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 AAV. For adherent HEK293, an AAV titer in the range of 1x 10⁹-1x10¹⁰ vp/mL and full to empty capsid ratio of 15-20 % were obtained from the cell culture study. 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. In this presentation, challenges associated with 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. Use of AC for AAV purification followed by AEXC for enrichment of full capsids will be elaborated with experimental results under different operating conditions. In addition, the critical role of UF/DF for protein clearance and intermediate formulation for mitigating aggregate formation will be elucidated. 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 purification will be discussed. This study was performed at KGI in collaboration with Celltheon Corp.