Development of a scalable and robust AEX method for enriched rAAV preparations in genome-containing VCs of serotypes 5, 6, 8, and 9

Abstract

Removal of empty capsids from adeno-associated virus (AAV) manufacturing lots remains a critical step in the downstream processing of AAV clinical-grade batches. Because of similar physico-chemical characteristics, the AAV capsid populations totally lacking or containing partial viral DNA are difficult to separate from the desired vector capsid populations. Based on minute differences in density, ultracentrifugation remains the most effective separation method and has been extensively used at small scale but has limitations associated with availabilities and operational complexities in large-scale processing. In this paper, we report a scalable, robust, and versatile anion-exchange chromatography (AEX) method for removing empty capsids and subsequent enrichment of vectors of AAV serotypes 5, 6, 8, and 9. On average, AEX resulted in about 9-fold enrichment of AAV5 in a single step containing 80% ± 5% genome-containing vector capsids, as verified and quantified by analytical ultracentrifugation. The optimized process was further validated using AAV6, AAV8, and AAV9, resulting in over 90% vector enrichment. The AEX process showed comparable results not only for vectors with different transgenes of different sizes but also for AEX runs under different geometries of chromatographic media. The herein-reported sulfate-salt-based AEX process can be adapted to different AAV serotypes by appropriately adjusting elution conditions to achieve enriched vector preparations.

Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors

“…In particular, when we applied a step protocol for buffer B, we achieved an almost baseline separation of the full rAAV and the empty capsid peaks. Other studies have also achieved full/empty separation for different AAV serotypes by AEX [ 8 , 24 , 34 , 37 , 38 , 39 , 40 ]. For determination of the degree of separation of full rAAVs from empty capsids, we used analytical AEX.…”

Retention and Fouling during Nanoparticle Filtration: Implications for Membrane Purification of Biotherapeutics

“…Density gradient centrifugation allows for the effective purification of viral capsids on a small scale, but this technique is difficult to apply for large-scale processing [ 11 ]. Size-exclusion (SEC) and ion-exchange (IEX) chromatography have both been explored for virus purification, but the large size of the nanoparticles leads to low binding capacities and significant mass transfer limitations [ 12 , 13 ].…”

High-efficiency purification of divergent AAV serotypes using AAVX affinity chromatography

“…While we did not explore this in the present work, multiple different downstream steps to enrich for full capsids utilizing size exclusion, anion exchange, or other chromatographic methods have been recently reported. 10 , 11 , 13 , 29 , 30 , 31 , 32 , 33 , 34 , 35 These can be added in series as additional steps to the process after the AAVX affinity binding step.…”