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Study of Requirements for Quality Control of Recombinant Adenovirus-Mediated Human Endostatin
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Abstract:
Objective: To establish the quality control methods and requirements for recombinant adenovirus-human endostatin products. Methods: E2B region on the adenovirus vector and inserted endostatin gene were identified by PCR. The restricted enzyme digestive map of recombinant virus DNA was analysed by agarose gel eletrophoresis. The number of virus particle and infectious titer were determined by UV and TCID50 method respectively. The expression level of inserted gene was analysed by infection of human HepG2 cell. The potency of expression products was determined by its inhibition effects on the proliferation of human HM2 endothelial cell. The purity of Adv-endostatin was analysed by UV and IE-HPLC. The replication competent virus was detected by A549 cell method. Results: The PCR results of E2B region and endostatin gene on the vector were conformed to theoretics. The restricted enzyme digestive map of detected recombinant virus DNA was identical to that of the reference. The number of virus particle, the infectious titer and the ratio of infectious titer to virus particle for the bulk were 2.4×10 12 VP/ml, 1.53×10 11 IU/ml, and 6.4% IU/VP respectively. The number of virus particle, the infectious titer and the ratio of infectious titer to virus particle for the finished product were 1.0×10 12 VP/ml, 3.75×10 11 IU/ml and 3.8% IU/VP respectively. After the HepG2 cell was infected by recombinant virus for 48 hours, the concentration of endostatin in the culture was 332 ng/ml. The inhibition rate of 50 MOI recombinant adenovirus to endothelial cell was 55%. The A260nm/A280nm ratio was 1.29. The purity determined by IE-HPLC was 99.7%. There were less than one replication competent virus within 3×10 10 VP recombinant adenovirus products. Other items complied with its corresponding requirements. Conclusion: The methods and requirements had been established for quality control of Adv-human endostatin.
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