Quality control methods for adenovirus CNHK200-hEndostatin / 第二军医大学学报

ABSTRACT

Objective: To establish a quality control method for Gene-Viral Therapy system CNHK200-hEndostatin. Methods: According to "The Guideline on Quality Control Methods of Human Gene Therapy Products", a quality control method for adenovirus was set up, which consisted of adenovirus protein identification, genomic identification, hEndostatin gene identification, hEndostatin protein quantitation, adenovirus particle quantitation, virus titer quantitation, determination of particle to PFU ratio, purity detection, AdWT detection, host cell DNA residue detectiong bovine serum protein residue detection, etc. The purified adenovirus product of CNHK200-hEndostatin was subjected to the above process. Results: We set up a SDS-PAGE method to identify the adenovirus protein and identified adenovirus genomic DNA by restriction endonuclease enzyme analysis. Human endostatin gene was identified by PCR method and its expression product was quantitated by ELISA. The number of adenovirus particle was quantitated by D260 method and HPLC method. The purity of adenovirus was determined by D260/D280 method and HPLC method. A PCR reaction was introduced to detect AdWT and hybridization method was used in host cell DNA residue detection. Bovine serum protein residue was detected by reverse indirect hemagglutination assay. All these methods were confirmed feasible in adenovirus quality control and our purified adenovirus sample was eligible in all test items. Conclusion: A series of basic methods have been successfully established for quality control of gene-viral therapy system CNHK200-hEndostatin. Our modified method for adenovirus particle quantitation by HPLC is more rapid and accurate than traditional method. The established methods have been approved in testing the purified adenovirus samples.