RESUMO
Adenovirus preparations are used as vectors in a number of gene therapy clinical development programs. The success of commercial production of adenovirus will strongly depend on the development of methods to define the recombinant virus product by analysis as opposed to being defined by the manufacturing process. While most analytical techniques examine portions of the virus, e.g. proteins or DNA, ion-exchange chromatography has been used to separate intact virus at low efficiency. A free zone capillary electrophoretic method was developed for high-efficiency separations of adenovirus 5. Experimental conditions such as buffer pH and concentration were explored which produced a high-efficiency separation in less than 20 min. The virus band was identified by collection of CE fractions and examination using a cell based assay. Initially, a single virus peak is found in fresh virus samples. After as little as one freeze-thaw in 1 x phosphate-buffered saline with 2% sucrose, the active virus migrates as a regular series of peaks. The nature of the virus modification leading to the differing electrophoretic mobilities is presently under investigation.
Assuntos
Adenoviridae/isolamento & purificação , Eletroforese Capilar/métodos , Recombinação Genética , Adenoviridae/genética , Reação em Cadeia da PolimeraseAssuntos
Genoma , Proteínas/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de Proteína/métodos , Animais , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese Capilar/métodos , Fermentação , Terapia Genética/métodos , Vetores Genéticos/análise , Vetores Genéticos/genética , Humanos , Espectrometria de Massas/métodos , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , Proteínas/análise , Análise de Sequência de DNA/tendências , Análise de Sequência de Proteína/tendênciasRESUMO
An RP-HPLC assay was developed for a recombinant adenovirus type 5. During chromatography, intact adenovirus dissociated into its structural components (DNA and proteins) and the viral proteome was separated yielding a characteristic fingerprint. The individual components were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, N-terminal sequencing and amino acid composition. The assay was utilized to measure adenovirus particle concentration through quantification of structural proteins. Each structural protein provided independent measurement of virus concentration allowing verification of accuracy. The assay sensitivity is at or below 2 x 10(8) particles. Contrary to the benchmark spectrophotometric assay, the RP-HPLC assay was shown to be insensitive to contaminants common for partially purified adenovirus preparations.
