Cation-exchange high-performance liquid chromatography of recombinant adeno-associated virus type 2.
There has been much interest recently in the development of recombinant viruses as vectors for gene therapy applications. We have constructed a recombinant adeno-associated viral (AAV) vector containing the gene encoding CFTR (cystic fibrosis transmembrane chloride regulator). This vector is currently being used in clinical trials as a treatment for cystic fibrosis. In the course of scale-up and process optimization efforts, a variety of analyses have been developed to characterize yield and quality. Although these methods produce quantitative and highly reproducible results, most are very time intensive. For example, a standard bioassay requires a 72-h incubation period followed by an additional day of analysis. Other tests such as UV spectrophotometry are fast, but unable to distinguish between whole virus, free protein, and DNA. Here, we describe an analytical cation-exchange high-performance liquid chromatographic method utilizing a TSKgel SP-NPR strong cation-exchange column. Unlike the bioassay which requires a 96-h wait for information, this method yields data in less than 20 min. In addition to the quick assay turn-around, the material eluting in the single peak was found to be intact, infectious, nuclease resistant AAV particles. This offers a significant advantage over the limited information one gains from UV spectrophotometry. This demonstrates the utility of chromatography for analysis and purification of viral vectors.
Debelak
D
D
Targeted Genetics Corporation, Seattle, WA 98101, USA.
Fisher
J
J
Iuliano
S
S
Sesholtz
D
D
Sloane
D L
DL
Atkinson
E M
EM
D015153:Blotting, Western; D002412:Cations / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D000229:Dependovirus / Q000737:chemistry* / Q000235:genetics; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000235:genetics; D012015:Reference Standards; D013056:Spectrophotometry, Ultraviolet
D002412:Cations; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator