Abstract:

Objective To develop a national reference of the freeze-dried recombinant Mycobacterium tuberculosis allergen for the bulk potency evaluation, in which the recombinant Mycobacterium tuberculosis fusion protein (EC) (Which is the Chinese generic name of the drug determined by the National Pharmacopoeia Committee, EC stands for recombinant fusion protein 6 kDa early secretory antigenic target (ESAT-6) and 10 kDa culture filtrate protein (CFP-10)) bulk was used as the material. Methods The EC bulk with qualified purity determined by electrophoresis and HPLC was weighed, packaged and freeze-dried accurately to make as national reference candidate, and the content of protein and moisture was further verified. The candidate of different dilution was injected into guinea pig infected with live Mycobacterium tuberculosis, a proper dilution was designated according to the reaction of skin test. And then the applicability of national reference candidate was evaluated preliminarily through the potency testing of the recombinant EC bulk. Stability of candidate storing in -20℃ for 24 months was also evaluated. Results The purity of EC stock was 100.00% by electrophoresis, and 95.33% by HPLC. The protein content of national reference candidate was 508μg/bottle, moisture content was 1.57%, and sub-packaging precision was controlled within ±1%. There was a good linear relationship in the dose response curve (R²=0.9944)when the dilution of reference candidate was 2.5 μg/ml (12.5 U/ml), 5 μg/ml (25 U/ml), and 10 μg/ml (50 U/ml). Assaying the national reference candidate by EC stock, the trend of both logarithmic reaction curve was corresponding (R²=0.9878,R²=0.9643), and ratio met 1.0±0.2. Method validation showed that the trend of logarithmic reaction curve of national reference candidate and EC bulk was corresponding (R²=0.9999,R²=0.9815), and ratio met 1.0±0.2. The result of stability study showed that national reference candidates were stable when they were kept at -20℃ for 24 months. Conclusion The candidate meets the demand of national reference in terms of purity, protein content, moisture content and uniformity. It will be possible to be used in the standization of potency assay of the freeze-dried recombinant Mycobacterium tuberculosis allergen.in a proper dilution. After method validation and stability evaluation, the candidate shows good performance, which will provide a good basis for the application of national reference

Key words: Mycobacterium tuberculosis, Recombinant fusion proteins, Freeze drying, Reference standards, National reference panel, Potency assay