Scalable Purification of Adeno-Associated Virus Type 2, 4, or 5 Using Ion-Exchange Chromatography

Abstract

The availability of high-titer, high-purity, adeno-associated virus type 2 (AAV2) stocks has dramatically increased our understanding of this virus and its utility as a gene transfer vector. Current methods of purification take advantage of the stable interaction of AAV2 with heparin sulfate. This affinity chromatography, however, is not useful for purifying AAV4 and AAV5, because these serotypes lack heparin-binding activity. We have developed simple ion exchange high-performance liquid chromatography (HPLC) method for purifying different AAV serotypes that does not rely on the affinity of the viruses for heparin. The protocol is fast, efficient, and yields highly infectious material. Analysis of the highly purified virus indicated that more than 90% of the particles contained genomes and were more active than virus purified by cesium chloride (CsCl) gradient purification. This procedure is scalable and can easily be streamlined for large-scale production of recombinant adeno-associated virus (rAAV), regardless of the serotype. Ultimately, the new purification method will further the characterization of rAAV of different serotypes as vectors for gene therapy applications.