Abstract:

Objective To establish the quality standard for 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)) stock solution and recombinant EC. Methods Six batches of recombinant EC stock solution, 11 batches of recombinant EC, 21 no specific pathogen (SPF) white guinea pigs with the weight of 300-400 g, viable BCG bacterial solution (1mg/ml), and viable bacteria solution of Mycobacterium tuberculosis attenuated strain H37Ra (2mg/ml) were selected to establish the quality standard of the recombinant EC stock solution, including residual antibiotic activity detection, sensitization effect test, potency determination and animal identification experiment. Establishment of quality standards for recombinant EC included identification experiment and potency determination. Six consecutive batches of the recombinant EC stock solution and 11 batches of the recombinant EC were tested using to the established quality standard, to verify the applicability and feasibility of the standard. Results 1. Data of the residual antibiotic activity detection in the recombinant EC stock solution: (1) Specific experiment: the absorbance (A) value of PBS buffer was 1.736, and that of kanamycin standard (0.0 ng/ml) was 2.178; (2) Linear and range experiments: the determination coefficient R² of goodness of fit of regression equation for three consecutive experiments was greater than 0.99 when kanamycin ranged between 0.5 and 40.5 ng/ml; (3) Accuracy test: the recovery rates of residual kanamycin in three consecutive batches of recombinant EC were 112.163%, 117.285% and 103.527%, respectively; (4) Repeatability test: the relative standard deviation (RSD) of A values of repeatability test in three consecutive batches of recombinant EC were 5.59%, 9.18% and 8.07%, respectively; (5) Intermediate precision test: kanamycin residues of three consecutive batches of the recombinant EC stock solution were detected by the same experimenter at different times and by different experimenters at the same time, and the results were all less than the quantitative limit of 0.5 ng/ml; (6) Durability test: the change of reading time (0, 5, 10min) had no effect on the detection of kanamycin residues in recombinant EC stock solution, and kanamycin residues were all less than the quantitative limit of 0.5 ng/ml. 2. Potency determination of recombinant EC stock solution: when the dilutions were 1μg/ml (5 U/ml), 2.5μg/ml (12.5 U/ml), 5μg/ml (25 U/ml) and 10μg/ml (50 U/ml), the average diameters and sum ratios of the average diameter of induration or redness of 2 batches of recombinant EC stock in animal experiments were (15.17±0.88) mm, (13.67±1.25) mm, 1.11 (91.00/82.00); (17.00±1.76) mm, (16.08±1.32) mm, 1.06 (102.00/96.50); (19.58±1.69) mm, (17.67±1.37) mm, 1.11 (117.50/106.00); (20.75±1.57) mm, (20.25±1.17) mm, 1.02 (124.50/121.50), respectively. 3. Animal identification experiment for recombinant EC stock solution: the average diameters of induration or redness in 3 consecutive batches of recombinant EC were 0, while the average diameter of induration was (15.13±5.06) mm in TB-PPD. 4. Identification test of the recombinant EC: the average diameters of induration or redness in 3 consecutive batches of recombinant EC were 0, while the average diameter of induration was (16.50±2.65) mm in TB-PPD. 5. Quality standard verification: (1) Recombinant EC stock solution: the results of residual antibiotic activity were all lower than the quantitative limit of 0.5 ng/ml; the sensitization effect test results showed that there was no significant difference in the local reactions of the injection site and no systemic reaction were found; the results of potency determination showed that in different dilutions (2.5μg/ml (12.5 U/ml), 5μg/ml (25 U/ml), 10μg/ml (50 U/ml)), the average diameter of local induration or redness reaction of each dilution were all ≥8 mm 24 h after injection. The ratio of the sum of local induration or redness reaction of the corresponding controls was 0.9±0.1. Results of animal identification experiment showed that the skin tests of BCG viable bacteria sensitized guinea pigs were all negative (mean diameter of induration or redness <5 mm), while the skin tests of guinea pigs with TB-PPD were positive (mean induration diameter ≥5 mm); (2)Tests for the recombinant EC: the results showed that the skin test were all negative (mean induration or redness diameter <5 mm), while TB-PPD were positive (mean induration diameter ≥5 mm). Conclusion The established quality standards could be used for verification in recombinant EC stock solution and recombinant EC.

Key words: Recombinant Mycobacterium tuberculosis fusion protein (EC), Quality control, Reference values, Drug evaluation, In vitro