ADENOVIRUS-ASSOCIATED VIRUSES SEPARATION METHOD
Provided is a method of enriching full adenovirus-associated virus (AAV) capsids from a mixture of full AAV capsids and empty AAV capsids, the method comprising providing a sample comprising a mixture of full AAV capsids and empty AAV capsids, subjecting the sample to anion exchange chromatography comprising an elution buffer comprising an equilibration buffer and a salt-containing buffer in an initial ratio that provides an initial conductivity, and changing the ratio of the equilibration buffer and the salt-containing buffer to provide a step gradient conductivity increase of about 0.5-2.0 mS/cm in each step to elute empty AAV capsids and to provide a fluid enriched with full AAV capsids.
1 . A method of enriching full adenovirus-associated virus (AAV) capsids from a mixture of full AAV capsids and empty AAV capsids, the method comprising
providing a sample comprising a mixture of full AAV capsids and empty AAV capsids,
subjecting the sample to anion exchange chromatography comprising an elution buffer comprising an equilibration buffer and a salt-containing buffer in an initial ratio that provides an initial conductivity in the range of about 0.5 mS/cm to about 10 mS/cm, and
changing the ratio of the equilibration buffer and the salt-containing buffer to provide a step gradient conductivity increase of about 0.5-2.0 mS/cm in each step to elute empty AAV capsids and to provide a fluid enriched with full AAV capsids.
2 . The method of claim 1 , wherein subjecting the sample to anion exchange chromatography comprises contacting a positively charged microporous membrane with the sample.
3 . The method of claim 1 , wherein a final step provides a conductivity of about 20 mS/cm.
4 . The method of claim 1 , wherein the initial ratio of equilibration buffer to salt-containing buffer is about 98.3:1.7.
5 . The method of claim 1 , wherein the final ratio of equilibration buffer to salt-containing buffer is about 81.5:18.5.
6 . The method of claim 2 , wherein a final step provides a conductivity of about 20 mS/cm.
7 . The method of claim 2 , wherein the initial ratio of equilibration buffer to salt-containing buffer is about 98.3:1.7.
8 . The method of claim 3 , wherein the initial ratio of equilibration buffer to salt-containing buffer is about 98.3:1.7.
9 . The method of claim 2 , wherein the final ratio of equilibration buffer to salt-containing buffer is about 81.5:18.5.
10 . The method of claim 3 , wherein the final ratio of equilibration buffer to salt-containing buffer is about 81.5:18.5.
11 . The method of claim 4 , wherein the final ratio of equilibration buffer to salt-containing buffer is about 81.5:18.5.