Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield.
Conventional methods for rAAV purification that are based on cesium chloride ultracentrifugation have often produced vector preparations of variable quality and resulted in significant loss of particle infectivity. We report here several novel purification strategies that involve the use of non-ionic iodixanol gradients followed by ion exchange or heparin affinity chromatography by either conventional or HPLC columns. These methods result in more than 50% recovery of rAAV from a crude lysate and routinely produce vector that is more than 99% pure. More importantly, the new purification procedures consistently produce rAAV stocks with particle-to-infectivity ratios of less than 100, which is significantly better than conventional methods. The new protocol increases the overall yield of infectious rAAV by at least 10-fold and allows for the complete purification of rAAV in 1 working day. Several of these methods should also be useful for large-scale production.
Zolotukhin
S
S
Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA.
Byrne
B J
BJ
Mason
E
E
Zolotukhin
I
I
Potter
M
M
Chesnut
K
K
Summerford
C
C
Samulski
R J
RJ
Muzyczka
N
N
D015153:Blotting, Western; D002499:Centrifugation, Density Gradient / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D000229:Dependovirus / Q000302:isolation & purification*; D005822:Genetic Vectors; D006493:Heparin / Q000378:metabolism; D006801:Humans; D014283:Triiodobenzoic Acids / Q000378:metabolism*
D014283:Triiodobenzoic Acids; D006493:Heparin; C044834:iodixanol