Introduction: Recombinant adenovirus preparations are used for gene delivery in a growing number of clinical development programs in gene therapy. In support of the anticipated commercial scale production of recombinant adenovirus, analytical tools have to be developed to define the virus product.
Methods: In this study we employed different liquid chromatography methods in combination with mass spectroscopy to analyze the proteome of a recombinant adenovirus type 5. The methods include separation of adenoviral proteins by reverse-phase high-performance liquid chromatography followed by tryptic digestion and analysis by LC/MS. Alternatively the purified whole virus was digested with trypsin and the peptides were separated either by one dimensional reverse-phase chromatography or by two-dimensional liquid chromatography and analyzed by tandem mass spectroscopy.
Preliminary Data: A total of 11 protein species could be identified with this approach. This set included all major viral proteins and two minor proteins (viral endopeptidase and late L1 protein), which were identified for the first time by chromatography based assays. The detection of the viral endopeptidase, which is known to be expressed at or below 10 copies per virus, demonstrates the high sensitivity of our method. The methods reported in this study describe very sensitive analytical tools that can be applied to define the virus product by analysis.
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