Abstract:

Objective To investigate the stability of the bulk storage, finished products storage; in-use stability; and efficacy of recombinant Mycobacterium tuberculosis fusion protein(EC)(Chinese generic name of the drug determined by the National Pharmacopoeia Committee, EC stands for recombinant fusion protein 6-kDa (Relative molecular mass of 1 kDa is 1000) early secreted antigenic target protein (ESAT-6) and the 10-kDa culture filtrate protein (CFP-10))(Hereinafter referred to as “EC”). Methods (1) Bulk storage stability study:3 batches of bulk was placed at -70℃ and observed for 0-36 months, samples were taken in the 0, 3, 6, 9, 12, 18, 24, 36 months for sensitization effect test, identification test, titer test and tests of isoelectric point, molecular weight, protein content, purity (Electrophoresis), etc. In the sensitization effect test, guinea pigs were divided into experimental group (Injection of EC bulk) and control group (Injection of stabilizer), and the animal reaction after injection was observed, where there should be no difference between the two groups. In identification test, BCG sensitized guinea pigs were intradermally injected with EC and tuberculin pure protein derivative(TB-PPD) respectively. The skin test results of guinea pigs were observed and measured. The skin test results of EC should be negative (The average diameter of induration or redness <5 mm), while the skin test results of TB-PPD should be positive (The average diameter of induration ≥5 mm). In titer tests, 6 MTB sensitized guinea pigs, each was injected with 3 EC injections in different dilutions and 3 control injections, observe and measure the test results of guinea pig skin tests, the ratios of the sum of the average diameter of induration or redness of the local reaction to EC bulk injections in different dilutions and that to control injections, should be 1.0±0.2. The isoelectric points should be 3.5-5.3; the molecular weights should be (23±2.0) kDa; the protein contents should be not less than 450.0μg/ml, and the purities (electrophoresis method) should not be less than 95.0%. (2) Final products storage stability study: The final products were stored at 37℃ and 2-8℃ for observed 0-28 days and 0-36 months, respectively. On day 0, 7, 14, 21, 28 at 37℃ and month 0, 3, 6, 9, 12, 18, 24, 36 at 2-8℃, samples were taken for identification test, titer test and pH value, phenol content, etc. For the identification test, test method and result determination see ‘EC bulk’. For titer test, 4 guinea pigs, each was injected with EC final products and control injection respectively, the measurement methods, and the ratios of the sum of the average diameter determination see ‘EC bulk’, pH values should be 6.8-7.4, phenol contents should not be higher than 3.0g/L. (3) Final products in-use stability study: After opening, the final products were placed at two conditions: (25±2)℃, (60±5) % humidity, and (40±2℃), (75±5) % humidity, observe for 50min, to investigate the in use stability, samples were taken at 0, 10,20,30,40,50min, for identification test, titer test and pH value, osmotic pressure molar concentration, test methods and result determination see ‘EC final products’, osmotic pressure molar concentrations should be (280.0±68.0) mOsmol/kg. The stability of the products will be judged according to whether the regular sampling test results meet EC quality standards, it will provide basis for the determination of the validity period of EC bulk and final products, and the in-use effective period of EC final products. Results (1) For EC bulk stored at -70℃, observed for 0-36 months: At each time point, the sensitization effect tests showed no difference between the experimental group and the control group. The identification test results were negative for EC bulk and positive for TB-PPD; for titer test, the ratios of the sum of the average diameter of induration or redness of EC bulk and control injections were 0.9-1.1. The isoelectric points were 3.7-5.2. The molecular weights were (23.2-24.2) kDa. The protein contents were (501.6-616.7)μg/ml, and the purities (electrophoresis method) was 100.0%. (2) For EC final products stability study: At each time point, the identification test results were negative for EC bulk and positive for TB-PPD. For titer test, the ratios of the sum of the average diameter of induration or redness of EC final products and control injections were 0.9-1.0. pH values were 7.1-7.4 and phenol contents were (2.6-2.9) g/L. (3) For final products in-use stability study: At each time point, the identification test results were negative for EC bulk and positive for TB-PPD. For titer test, the ratios of the sum of the average diameter of induration or redness of EC final products and control injections were all 1.0. pH values were 7.1-7.2. Osmotic pressure molar concentrations were (302.4-307.4)mOsmol/kg. Conclusion EC bulk is quality stable to be stored for 36 months at -70℃. EC final products are quality stable to be stored for 28 d at 37℃ and 36 months at 2-8℃. After opening, the final products are quality stable for 50min.

Key words: Recombinant Mycobacterium tuberculosis fusion protein(EC), Drug stability, Efficacy study, Drug evaluation