title: Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography.
abstract: Adeno-associated virus (AAV) empty capsids typically co-purify with genome containing AAV2 vectors purified by column chromatography. This study describes a method to remove empty capsids from genome containing vector particles by anion exchange chromatography. The separation is based on the slightly less anionic character of empty particles compared to vectors. Detailed methods to achieve AAV2 vector purification and particle separation using cation exchange resin POROS 50HS followed by anion exchange resin Q-Sepharose(xl) are described. Chromatographic separation of AAV2 particles was achieved using gradients based on sodium acetate and ammonium acetate, and was optimal at pH 8.5. Efficient removal of particle surface nucleic acid impurities was found to be important to achieve good particle separation. In a large scale experiment performed using partially purified vector containing a mixture of 1.56 x 10(14)vg and 2.52 x 10(15) empty capsids as a starting material, the optimized anion exchange chromatography method resulted in a vector peak of 1.15 x 10(14)vg containing 0.25 x 10(14) empty capsids, corresponding to 74% vector yield and 86-fold reduction in empty capsids in the vector product.
authors.affiliation: Avigen Inc., Alameda, CA 94502, USA.
authors.forename: Guang
authors.initials: G
authors.lastname: Qu
authors.forename: Jennifer
authors.initials: J
authors.lastname: Bahr-Davidson
authors.forename: Joseph
authors.initials: J
authors.lastname: Prado
authors.forename: Alex
authors.initials: A
authors.lastname: Tai
authors.forename: Floro
authors.initials: F
authors.lastname: Cataniag
authors.forename: Jennifer
authors.initials: J
authors.lastname: McDonnell
authors.forename: Jingmin
authors.initials: J
authors.lastname: Zhou
authors.forename: Bernd
authors.initials: B
authors.lastname: Hauck
authors.forename: Jac
authors.initials: J
authors.lastname: Luna
authors.forename: Jurg M
authors.initials: JM
authors.lastname: Sommer
authors.forename: Peter
authors.initials: P
authors.lastname: Smith
authors.forename: Shangzhen
authors.initials: S
authors.lastname: Zhou
authors.forename: Peter
authors.initials: P
authors.lastname: Colosi
authors.forename: Katherine A
authors.initials: KA
authors.lastname: High
authors.forename: Glenn F
authors.initials: GF
authors.lastname: Pierce
authors.forename: J Fraser
authors.initials: JF
authors.lastname: Wright
mesh_terms: D002213:Capsid / Q000378:metabolism / Q000648:ultrastructure; D002852:Chromatography, Ion Exchange / Q000379:methods*; D000229:Dependovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005822:Genetic Vectors / Q000302:isolation & purification* / Q000648:ultrastructure; D006863:Hydrogen-Ion Concentration; D014771:Virion / Q000648:ultrastructure