title: A simplified purification protocol for recombinant adeno-associated virus vectors.
abstract: We describe a new rapid, low cost, and scalable method for purification of various recombinant adeno-associated viruses (rAAVs) from the lysates of producer cells of either mammalian or insect origin. The method takes advantage of two general biochemical properties of all characterized AAV serotypes: (i) low isoelectric point of a capsid and (ii) relative biological stability of the viral particle in the acidic environment. A simple and rapid clarification of cell lysate toremove the bulk of proteins and DNA is accomplished by utilizing inexpensive off-the-shelf reagents such as sodium citrate and citric acid. After the low-speed centrifugation step, the supernatant is subjected to cation exchange chromatography via sulfopropyl (SP) column. The eluted virus may then be further concentrated by either centrifugal spin devices or tangential flow filtration yielding material of high titer and Good Manufacturing Practice (GMP) grade biochemical purity. The protocol is validated for rAAV serotypes 2, 8, and 9. The described method makes rAAV vector technology readily available for the low budget research laboratories and could be easily adapted for a large scale GMP production format.
authors.affiliation: Powell Gene Therapy Center , Gainesville, Florida, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Potter
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for Structural Biology, University of Florida , Gainesville, Florida, USA.
authors.forename: Bridget
authors.initials: B
authors.lastname: Lins
authors.affiliation: Institute of Virology, Charité Medical School , Berlin, Germany.
authors.forename: Mario
authors.initials: M
authors.lastname: Mietzsch
authors.affiliation: Institute of Virology, Charité Medical School , Berlin, Germany.
authors.forename: Regine
authors.initials: R
authors.lastname: Heilbronn
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for Structural Biology, University of Florida , Gainesville, Florida, USA.
authors.forename: Kim
authors.initials: K
authors.lastname: Van Vliet
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for Structural Biology, University of Florida , Gainesville, Florida, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Chipman
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for Structural Biology, University of Florida , Gainesville, Florida, USA.
authors.forename: Mavis
authors.initials: M
authors.lastname: Agbandje-McKenna
authors.affiliation: Powell Gene Therapy Center , Gainesville, Florida, USA.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Cleaver
authors.affiliation: Powell Gene Therapy Center , Gainesville, Florida, USA ; Department of Pediatrics, University of Florida , Gainesville, Florida, USA .
authors.forename: Nathalie
authors.initials: N
authors.lastname: Clément
authors.affiliation: Powell Gene Therapy Center , Gainesville, Florida, USA ; Department of Pediatrics, University of Florida , Gainesville, Florida, USA .
authors.forename: Barry J
authors.initials: BJ
authors.lastname: Byrne
authors.affiliation: Powell Gene Therapy Center , Gainesville, Florida, USA ; Department of Pediatrics, University of Florida , Gainesville, Florida, USA .
authors.forename: Sergei
authors.initials: S
authors.lastname: Zolotukhin