» BCG Live (intravesical) for immunotherapy is a freeze-dried preparation of attenuated live bacteria derived from a culture of Bacillus Calmette-Guérin (Mycobacterium bovis, var. BCG) and used intravesically in the treatment of carcinoma in situ and papilloma tumors of the urinary bladder. The bacteria are grown in a medium that does not contain substances known to cause toxic or allergic reactions in human beings or to cause the bacteria to become virulent for guinea pigs. The culture is harvested and formulated to contain one or more excipients. The freeze-dried preparation is reconstituted and further diluted aseptically with a sterile diluent for use. A reconstituted dose contains 1.019.2 × 108 colony-forming units (cfu). BCG Live does not contain a preservative.
Packaging and storage BCG Live is sensitive to light and therefore must be preserved and stored in a glass container where it is protected from direct light at a temperature between 2 and 8.
Expiration date The product is stable for 3 years when stored between 2 and 8.
Labeling Label it to indicate the dry weight of bacteria in a vial, cfu per dose, the storage conditions, the expiration date, and that it is not to be used after the expiration date given on the package. Label it to state that it should be protected from light and that it should be used immediately after reconstitution/dilution. Label it to indicate that it is for intravesical use.
Identification BCG Live is identified by microscopic examination of the bacilli in stained smears demonstrating their acid-fast property. Alternatively, validated molecular biology techniques may be used.
General safety It meets the requirements as set forth for Safety TestsBiologicals under Biological Reactivity Tests, In Vivo 88, modified as follows. Guinea pigs are injected intraperitoneally with 3.0 mL of the reconstituted product.
Sterility 71 It meets the requirements when tested as directed for Direct Inoculation of the Culture Medium under Test for Sterility of the Product to be Examined.
Test suspension Reconstitute the freeze-dried BCG Live as per the manufacturer's instructions for human use with the diluent recommended by the manufacturer, and dilute aseptically to about 2 mg per mL with sterile BCG diluent.
Procedure Randomly select not fewer than six guinea pigs of the same sex, each weighing 250 to 300 g. Inject each animal with a total of at least 4 mg of the Test suspension intramuscularly or subcutaneously in the rear left internal thigh, and observe them for a period of 6 weeks. Note the number of animals that survive at the end of the observation period, and then sacrifice them. Perform autopsies of all animals postmortem to examine them for evidence of tuberculous infections, particularly at the popliteal and inguinal lymph nodes, liver, spleen, pancreas, and lungs, as well as at the injection site. If any abnormalities are found, perform a histological examination using standard and Acid-Fast staining techniques to detect Acid-Fast organisms. The product complies with the test if none of the animals show signs of tuberculosis and not more than one-third of the animals die during the observation period.
Test suspensions Using the same diluent and the Test suspension prepared as directed in the test for Virulent mycobacteria, further dilute aseptically by making three serial tenfold dilutions.
Procedure Randomly select two guinea pigs (male or female), each weighing 250 to 300 g. Inject 0.1 mL of each of the four suspensions intradermally at different sites on the back of each animal. After 4 weeks, the animals are shaved so that the injection sites and any reactions are made clearly visible. The diameters of the reactions are measured, and the presence of necrosis or nodules are noted. The reaction for the largest dose is between 4 and 10 mm and the smallest dose induces a nodule less than or equal to 4 mm. Each animal gains weight during the observation period.
Tuberculin solution Use tuberculin, purified protein derivative, to prepare a solution containing 25 U.S. Tuberculin Units per 0.1 mL. Dilute aseptically, if necessary, with sterile 0.9% sodium chloride solution.
Procedure Use the same animals on which the Skin reactivity test is performed. After the Skin reactivity test is completed, inject each animal intradermally on the back with 0.1 mL of the Tuberculin solution, and observe after 18 to 24 hours. An erythematous reaction of not less than 10 mm in diameter is measured on each animal.
Residual moisture: Not greater than the limit approved for the particular product, determined by a suitable validated method. Limits vary in accordance with the method.
Viability Determine the potencies of BCG Live using not less than 5 containers before freeze-drying and an equal number of containers after freeze-drying, following the procedure described under Potency, except use the suspension before freeze-drying as is. The loss in viability due to freeze-drying is not more than 90%.
Potency Determine the number of viable units per mL by viable count on solid medium using a method suitable for the product to be examined. Alternatively, a validated biochemical method may be used.
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USP32NF27 Page 1630Pharmacopeial Forum: Volume No. 30(2) Page 452