Combining steric exclusion with anion exchange - development of a universal and scalable adeno-associated virus downstream process.

Adeno-associated viruses (AAV) are among the leading vectors for in vivo gene therapy. The purification of AAV remains a bottleneck as it typically requires multiple individual process steps, often resulting in product loss and high costs. Current downstream processes are usually serotype-specific and rely primarily on expensive affinity resins. To address these limitations, we developed a serotype-independent purification method using steric exclusion chromatography (SXC) that can be combined with a subsequent anion exchange full/empty separation step. This alternative approach eliminates the need for intermediate concentration and buffer exchange, thereby reducing the number of process steps required while achieving high-purity full AAV particles. SXC conditions were optimized using a design of experiments approach. Isocratic separation of full and empty AAV resulted in further purification of the sample. The overall process achieved a viral genome recovery of 51.7 %, along with impurity depletions of 99.9 % for DNA and 99.8 % for protein. The process was successfully adapted to different AAV serotypes and genes of interest, demonstrating its robustness and versatility. In addition, the scalability of SXC was demonstrated, highlighting its potential for large-scale manufacturing. This streamlined, universal, and scalable process provides a robust and efficient alternative to traditional AAV purification processes, addressing critical challenges in gene therapy production and paving the way for broader implementation in research and manufacturing.