A new scalable method for the purification of recombinant adenovirus vectors.
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.
Green
Anthony P
AP
Puresyn, Inc., Malvern, PA 19355, USA. tony@puresyn.com
Huang
James J
JJ
Scott
Miller O
MO
Kierstead
Timothy D
TD
Beaupré
Irene
I
Gao
Guang-Ping
GP
Wilson
James M
JM
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