title: Scaleable chromatographic purification process for recombinant adeno-associated virus (rAAV).
abstract: BACKGROUND
  Adeno-associated virus (AAV) is a human parvovirus currently being developed as a vector for gene therapy applications. Traditionally AAV has been purified from cell lysates using CsCl gradients; this approach however is not likely to be useful in large-scale manufacturing. Moreover gradient-purified AAV vectors tend to be contaminated with significant levels of cellular and adenoviral proteins and nucleic acid. To address the issue of purification we have developed a process scale method for the rapid and efficient purification of recombinant AAV (rAAV) from crude cellular lysates.
  
  
  METHODS
  The preferred method for the purification of rAAVbetagal includes treatment of virally infected cell lysates with both trypsin and nuclease followed by ion exchange chromatography using ceramic hydroxyapatite and DEAE-Sepharose in combination with cellufine sulphate affinity chromatography.
  
  
  RESULTS
  Purification of rAAV particles from crude cellular lysates co-infected with adenovirus was achieved using column chromatography exclusively. Column-purified rAAV was shown to be greater than 90% pure, free of any detectable contaminating adenovirus, biologically active, and capable of directing efficient gene transfer to the lungs of both cotton rats and mice.
  
  
  CONCLUSIONS
  This study demonstrates the feasibility of using column chromatography alone for the isolation of highly purified rAAV vector. The methods described here are advancements in procedures to purify rAAV and are adaptable for commercial production of clinical-grade rAAV vector.
authors.affiliation: Genzyme Corporation, Framingham, MA 01701-9322, USA. coriordan@genzyme.com
authors.forename: C R
authors.initials: CR
authors.lastname: O'Riordan
authors.forename: A L
authors.initials: AL
authors.lastname: Lachapelle
authors.forename: K A
authors.initials: KA
authors.lastname: Vincent
authors.forename: S C
authors.initials: SC
authors.lastname: Wadsworth
mesh_terms: D000818:Animals; D002460:Cell Line; D002845:Chromatography / Q000379:methods*; D002852:Chromatography, Ion Exchange; D004274:DNA, Recombinant / Q000302:isolation & purification; D000229:Dependovirus / Q000235:genetics / Q000302:isolation & purification*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008168:Lung / Q000378:metabolism / Q000821:virology; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016205:Sigmodontinae; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase