Chromatography purification of canine adenoviral vectors.
Canine adenovirus vectors (CAV2) are currently being evaluated for gene therapy, oncolytic virotherapy, and as vectors for recombinant vaccines. Despite the need for increasing volumes of purified CAV2 preparations for preclinical and clinical testing, their purification still relies on the use of conventional, scale-limited CsCl ultracentrifugation techniques. A complete downstream processing strategy for CAV2 vectors based on membrane filtration and chromatography is reported here. Microfiltration and ultra/diafiltration are selected for clarification and concentration of crude viral stocks containing both intracellular and extracellular CAV2 particles. A DNase digestion step is introduced between ultrafiltration and diafiltration operations. At these early stages, concentration of vector stocks with good recovery of viral particles (above 80%) and removal of a substantial amount of protein and nucleic acid contaminants is achieved. The ability of various chromatography techniques to isolate CAV2 particles was evaluated. Hydrophobic interaction chromatography using a Fractogel propyl tentacle resin was selected as a first chromatography step, because it allows removal of the bulk of contaminating proteins with high CAV2 yields (88%). An anion-exchange chromatography step using monolithic supports is further introduced to remove the remaining contaminants with good recovery of CAV2 particles (58-69%). The main CAV2 viral structural components are visualized in purified preparations by electrophoresis analyses. Purified vector stocks contained intact icosahedral viral particles, low contamination with empty viral capsids (10%), and an acceptable total-to-infectious particle ratio (below 30). The downstream processing strategy that was developed allows preparation of large volumes of high-quality CAV2 stocks.
Center of Animal Biotechnology and Gene Therapy (CBATEG), Universitat Autònoma de Barcelona, Barcelona 08193, Spain. mersegura@gmail.com
María Mercedes
MM
Segura
Meritxell
M
Puig
Mercè
M
Monfar
Miguel
M
Chillón
D006516:Adenoviruses, Canine / Q000235:genetics* / Q000648:ultrastructure; D000818:Animals; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004285:Dogs; D004586:Electrophoresis; D005374:Filtration / Q000379:methods; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D016133:Polymerase Chain Reaction; D013053:Spectrophotometry; D014771:Virion / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure