Abstract
Adeno-associated virus (AAV) vectors have been commonly purified through density gradient ultracentrifugation (DGUC) or column chromatography methods. Although the DGUC method can efficiently separate the empty from the full virus particles, its application in large-scale AAV purification is hindered due to its limitation in volume of each centrifuge tube. Alternatively, column chromatography is serotype-dependent, expensive, and complicated, which co-purifies both empty and full virus particles. In this study, we describe an economical and universal process using three-phase partitioning (TPP) combined with DGUC to purify large quantities of AAV vectors. First, TPP is used to remove up to 90% of the cellular impurities in the cell lysate and at the same time condense the AAV vectors into ∼10% of their original lysate volume. Second, two rounds of DGUC are employed to separate the empty from the full virus particles and at the same time remove the remaining cellular impurities. This combined process increases the capacity of ultracentrifugation by a factor of 5- to 10-fold depending on the yields of AAV serotypes. A variety of AAV serotypes such as AAV2, AAV5, AAV6, AAV9, and AAVDJ have been successfully purified with this process. Both in vitro and in vivo studies demonstrate that TPP has no detrimental impact on AAV infectivity. In a proof of concept, we performed several purification runs ranging from 3 to 25 L of Sf9 culture volume. We were able to purify more than 3e+15 viral genomes (vg) of AAV vectors from 3 L of cell culture volume with just two SW28 centrifuge tubes in a Beckman Coulter ultracentrifuge. Our data indicate that this TPP-DGUC process is economic, universal, and can be used to purify a large quantity of AAV vectors for clinical applications with just a few ultracentrifuges.
Highlights
Recombinant adeno-associated virus (AAV) vector has emerged as one of the most versatile and successful gene therapy delivery vehicles
Optimization of Ammonium Sulfate Concentration in the First Round of three-phase partitioning (TPP) In order to determine the optimal concentration of ammonium sulfate to salt-out AAV for purification, we tested a series of ammonium sulfate concentrations from 10% to 45%
Most AAV vectors remained in the L1 phase when 10% (a-L1), 15% (b-L1), or 20% ammonium sulfate was used
Summary
Recombinant adeno-associated virus (AAV) vector has emerged as one of the most versatile and successful gene therapy delivery vehicles. AAV purification with the traditional method through density gradient ultracentrifugation (DGUC) using either cesium chloride (CsCl) or iodixanol media is limited by the volume each centrifuge tube can hold. A 25-L production run would require 83 centrifuge tubes, or 14 ultracentrifuges, which is expensive and not practical. AAV purification with column chromatography is a preferred method in the industry, the process of column chromatography is serotypedependent, expensive, and complicated, and both empty and full AAV virus particles are usually co-purified. The AAV binding capacity of the media is generally in the range of 1e+12 viral genomes (vg)/mL, which translates into the requirement of a 100-L column for purification of 1e+17 vg of AAV vectors, and the cost of such a large column is in the millions of US dollars. It has been reported that polyethylene glycol (PEG)/aqueous two-phase partitioning methods could be used to purify AAV vectors, but the method yielded limited amounts of AAV vectors and the purities were not satisfactory.[7]
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