(157d) Development of the Purification Process for Gene Therapy Vector Adeno-Associated Virus (AAV)

As adeno-associated virus (AAV) vector engineering efforts continue to improve the transduction efficiency of specific organs, the AAV vector is becoming a more viable and popular alternative for gene therapy. AAV is highly inefficient at packaging its genome, with up to 90% of the formed AAV capsids lacking the gene of interest (GOI) packaged inside, commonly referred to as empty AAV. The upstream process often requires lysing the cells to release more viral particles to achieve a manufacturable viral titer, but cell lysis generates a significant amount of host cell impurities compared to therapeutic protein production processes. Additionally, engineered AAV capsids can be very labile, which creates further challenges for downstream processing. New resins, chromatography, membrane filtration modalities, and operational modes are being explored to improve purification processes with high yield, high doses, and high purity. Furthermore, the complex and variable physicochemical properties of these engineered AAV vectors necessitate the development of specific downstream processes.

Herein, a downstream purification process that can be applied to different AAV serotypes to streamline and accelerate manufacturing process development is explored. The process development emphasizes unit operations such as clarification, tangential flow filtration (TFF), affinity chromatography, and anion exchange (AEX) chromatography. An integrated AEX-based depth filter and a filter train comprising several traditional polypropylene, cellulose, and diatomaceous earth-based, as well as synthetic depth filters with minimal leachables, were screened to reduce host cells, cellular debris, particulates, and host cell impurities. The traditional depth filter train showed better throughput and a higher degree of reduction in host cell impurities and turbidity than the integrated filter. A large molecular weight cut-off (30-300 kDa MWCO) tangential flow filtration (TFF) cassette (mPES) and TFF capsule (regenerated cellulose) were compared to reduce the affinity load volume, smaller-sized impurities, and buffer exchange into the desired affinity column load buffer.

In addition to filtration steps, two chromatography steps are being developed. A large pore-sized affinity POROS AAVX resin was selected to capture AAV and reduce the majority of impurity burden while lowering the mass transfer diffusion hindrance caused by the large viral particles. A high virus recovery rate of over 90% was achieved for each serotype with optimized low pH step elution. An increase in resin loading was found to elevate virus recovery from the resin and improve step yield, consistent with peer publications. A new modality monolith AEX was compared with traditional resin-based AEX for the evaluation of full/empty capsid separation and further impurity polishing. More than a 40% increase in full AAV capsids was achieved using the optimal column and operating conditions. With these two steps of chromatography, impurities were greatly reduced to levels below the limit of detection in assays.

In summary, a scalable purification process, including harvest clarification, ultrafiltration/diafiltration, affinity chromatography for AAV capture, and AEX for AAV polishing, was developed for different serotypes with good performance.