Purification of adenoviral vectors using expanded bed chromatography

• Published in: Journal of virological methods Vol. 132; no. 1; pp. 121 - 126
• Main Authors: Peixoto, C., Ferreira, T.B., Carrondo, M.J.T., Cruz, P.E., Alves, P.M.
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 01-03-2006; Amsterdam Elsevier; New York, NY
• Subjects: Adenoviridae - genetics; Adenoviridae - growth & development; Adenoviridae - isolation & purification; Adenovirus; Biological and medical sciences; Blotting, Western; Cell Line; Chromatography, Ion Exchange - methods; DNA, Viral - analysis; Electrophoresis, Polyacrylamide Gel; Expanded bed chromatography; Fundamental and applied biological sciences. Psychology; Genetic Vectors - isolation & purification; Humans; Microbiology; Polymerase Chain Reaction; Purification; Scale-up; Techniques used in virology; Ultrafiltration; Viral Plaque Assay; Viral Proteins - analysis; Virology; Expanded bed chromatography; Purification; Adenovirus; Scale-up; Virus; Adenoviridae; Microbiology; Method; Vector; Chromatography; Virology
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2005.10.002

The increasing numbers of pre-clinical and clinical trials where recombinant adenoviral vectors are used for gene therapy and vaccination require the development of cost-effective and reproducible large scale purification strategies of the biologically active particles. Alternatives to the traditional laboratory scale CsCl density gradient ultracentrifugation method, such as fixed bed chromatography strategies, have been developed, but the yields of final recovery remain too low due mainly to the capture and concentration steps taking place before and between the chromatographic stages.
In this study, a rapid and efficient scaleable purification protocol allowing to obtain concentrated, pure and bioactive adenoviral vectors was developed. This allows efficient levels of binding to the column media and vector purification without centrifugation or filtration steps. Expanded bed chromatography followed by hollow fiber concentration allows the capture of viral particles directly from cellular extracts with high efficiency and vector purification is achieved in less than one working day with a minimal amount of sample handling, thus presenting an improvement over existing processes. The overall process yield reached 32%, representing an eight-fold improvement over results reported previously, while the purity is comparable to that obtained with the CsCl method.