Purification of adenoviral vectors using expanded bed chromatography.
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 scale-able 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.
Peixoto
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IBET/ITQB, Apartado 12, P-2780-901 Oeiras, Portugal.
Ferreira
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Carrondo
M J T
MJ
Cruz
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PE
Alves
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PM
D000256:Adenoviridae / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D015153:Blotting, Western; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004279:DNA, Viral / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction; D014462:Ultrafiltration; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000032:analysis
D004279:DNA, Viral; D014764:Viral Proteins