A new scalable method for the purification of recombinant adenovirus vectors

Hum Gene Ther. 2002 Nov 1;13(16):1921-34. doi: 10.1089/10430340260355338.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenoviridae / genetics
  • Adenoviridae / isolation & purification*
  • Blotting, Western
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • Electrophoresis, Polyacrylamide Gel
  • Gene Transfer Techniques*
  • Genetic Vectors*
  • Microscopy, Electron
  • Temperature