Manufacturing Process Intensification of Adeno-Associated Viral Vectors Type-8 Using Weak Partitioning Chromatography With UV-Based Process Analytical Technology (PAT).

Abstract

Separation of full and empty capsids is a critical step in manufacturing of recombinant adeno-associated viral vector (AAV) based gene therapies. Anion exchange chromatography (AEX) is well-established as a scalable method for full/empty separations. Due to the small differences in surface charge between full and empty capsids, shallow linear gradients of increasing conductivity are commonly used to resolve the two species. However, the resolution is adversely impacted by higher column loading, and most linear gradient processes load at 2e13-2e14 capsids/mL of monolith volume (cp/mL monolith) to achieve full capsid purity targets. With the rapid increase reported in upstream AAV titers up to > 1e12 vg/mL in the bioreactor over the last few years, current downstream purification processes utilizing linear gradient AEX are rapidly approaching scalability limits. This is particularly true for processes utilizing monoliths and membranes which are preferred over resins in AAV purifications due to elimination of pore size diffusion limitations. There is a pressing need for next-generation processes that can load more material in the range of > 1e15 cp/mL, while maintaining operating time and process robustness suitable for GMP manufacturing. This study presents a scalable method for empty/full separations for AAV8 using weak partitioning AEX combined with isocratic elution, demonstrated on CIM QA monoliths which are currently extensively used in the industry for AAV manufacturing. By optimizing load conditions and automating the process with UV-based signals, the strategy achieves > 80% full capsid purity and > 80% genomic yields. Compared to standard linear gradient AEX, the weak partitioning method achieves higher % full capsids as well as higher genomic yield due to combined effects of (i) removal of empty capsids in the flowthrough, (ii) reduction of nonspecific binding interactions as a result of column overloading, and (iii) elimination of peak-cutting by using isocratic elution. The approach enables > 10-fold higher loading per cycle while reducing processing time by 10-fold and is well-suited to rapid cycling or continuous processing operations to support next-generation, high-titer processes.