title: A new scalable method for the purification of recombinant adenovirus vectors.
abstract: Recombinant adenovirus vectors continue to be the preferred vectors for many types of gene therapy. However, issues regarding production and safety as well as the development of a scalable process for these vectors remain a challenge. Additionally, any process must address the well-documented immune and toxicologic responses to these vectors. Some alternatives to classic CsCl-gradient purification based on column chromatography have been developed, but these first-generation processes are still limited in potential application. We report the development of a tandem column chromatography process incorporating two resins; anion-exchange and PolyFlo (Puresyn, Inc., Malvern, PA). PolyFlo is used in a novel manner as a polishing step to remove additional host and viral proteins not removed by the anion-exchange capture step. By using the beta-galactosidase reporter vector, H5.CMV-lacZ, the purity of the product is improved compared to the same vector purified by 2x CsCl or anion-exchange alone as determined by high-performance liquid chromatography (HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; silver stain), Western analysis, electron microscopy, and particle:infectious (VP:IU) unit ratio. The recovery over the entire process is significantly better than 2x CsCl and higher than other first-generation tandem chromatography processes. This new process is reproducible and scalable to 10(15) input viral particles per run. Furthermore, the purified adenovirus product remains intact after multiple freeze/thaw cycles and is stable at 4 degrees C, -20 degrees C, and -75 degrees C. The process described here permits purification of adenovirus particles at a high concentration at large scale without centrifugation.
authors.affiliation: Puresyn, Inc., Malvern, PA 19355, USA. tony@puresyn.com
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Green
authors.forename: James J
authors.initials: JJ
authors.lastname: Huang
authors.forename: Miller O
authors.initials: MO
authors.lastname: Scott
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Kierstead
authors.forename: Irene
authors.initials: I
authors.lastname: Beaupré
authors.forename: Guang-Ping
authors.initials: GP
authors.lastname: Gao
authors.forename: James M
authors.initials: JM
authors.lastname: Wilson
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000302:isolation & purification*; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008854:Microscopy, Electron; D013696:Temperature