AAVpro Concentrator allows easy and efficient concentration of AAV vectors using a standard centrifuge. In contrast with other concentration methods that involve only precipitation of AAV particles, AAVpro Concentrator incorporates filtration steps to yield AAV preps of higher purity. The scalable mix-and-spin protocol can accommodate a range of input volumes and requires less than one hour of hands-on time. Figure 1 illustrates the workflow for preparing and purifying AAV vectors using AAVpro Concentrator.

Figure 1. An outline of the AAV vector enrichment process using AAVpro Concentrator.

Concentration of AAV vectors from serum-free culture supernatant

Culture supernatant containing AAV1 vectors expressing ZsGreen1 was concentrated from a starting input of 40 ml (one T225 flask) down to a final volume of 200 μl using AAVpro Concentrator. The viral titer was measured before and after concentration to determine the viral recovery rate using the AAVpro Titration Kit (for Real Time PCR) Ver.2.

Figure 2. Concentration and recovery rate of AAV1 vectors concentrated using AAVpro Concentrator.

Biological titer of concentrated AAV vectors

A biological assay commonly used to test the infectivity of an AAV preparation involves infecting permissive cells with AAV and measuring transgene expression to determine a titer in transducing units (TUs). To confirm the infectivity of AAV prepared using AAVpro Concentrator, flow cytometric analysis was used to compare ZsGreen expression in HT1080 cells infected with AAV1-ZsGreen1 vectors before or after concentration. HT1080 cells were infected with AAV at 5,000 or 500 vg/cell (MOI) and analyzed by FACS after three days. Concentrated AAV particles administered at MOIs of 5,000 or 500 were able to infect cells at rates of 44.7% or 9.2%, respectively.

Figure 3. AAV titration in transducing units, as determined by FACS analysis of ZsGreen1 expression in HT1080 cells.

AAVpro Concentrator can be used to concentrate AAV particles of any serotype from cell culture supernatants, including particles prepared using any AAV system from Takara Bio. Compared to ultracentrifugation, the user-friendly AAVpro Concentrator protocol is easier to execute, requires much less hands-on time, and can be performed using standard laboratory equipment. Processing of 40 ml of culture supernatant down to a final volume of 200 µl resulted in a 95-fold increase in AAV titer at a recovery rate of ~48%. The incorporation of filtration and ultrafiltration steps in the AAVpro Concentrator protocol allows for removal of impurities that may interfere with downstream applications. AAVpro Concentrator allows for processing of supernatants with or without serum, and can also be used to perform buffer exchanges.