USP Chapter <71> Sterility Test states that prior to conducting a sterility test on a sterilized product, its level of bacteriostatic and fungistatic activity should be determined, that is the degree of inhibition to microbial growth that may be caused by substances in or on the product. Bacteriostasis/Fungistasis (B/F) should be evaluated following the first sterilization or aseptic manufacturing of all new products prior to sterility testing or when significant product or manufacturing changes occur. The testing is designed to evaluate B/F activity and to validate the chosen sterility test method.
Based on the media type to be used in the Sterility Test, low numbers (<100 CFU) of selected bacteria and fungi challenge organisms as specified in the USP, are added to containers with the test article present to demonstrate that they can be detected in the presence of the test article.
For samples sterility tested by the membrane filtration method, test samples are filtered through a 0.45 µm membrane filter and rinsed with a sterile diluent. The rinse is then inoculated with the chosen organisms and the membrane filters are aseptically added to the appropriate media.
For samples sterility tested by the direct transfer method, test samples are aseptically transferred into a volume of media sufficient to cover the sample, the containers are then inoculated with the chosen organisms. The volume of media utilized in the B/F testing must be the same amount utilized for routine sterility testing procedures.
The B/F validation will demonstrate reproducibility of the method to reliably recover representative microorganisms. If growth is inhibited, a documented investigation must be initiated and modifications such as increased dilution, additional membrane filter washes or addition of inactivating agents should be implemented to the test method to optimize recovery.
Sterility testing is necessary for pharmaceuticals and medical devices that claim to be sterile or free from viable microorganisms. Sterility testing methods are required to be accurate and reproducible, in accordance with 21CFR 211.194 and 211.165. USP Chapter <71> Sterility Test is the principal source used for sterility testing of sterile drug products produced by aseptic processing. The quantity and number of products to be tested is determined both by batch size and individual container fill volumes. It is important that the samples represent the entire batch and processing conditions. Samples should be taken at the beginning, middle, and end of the aseptic processing operation.
The test evaluates samples for sterility by transferring the solution into growth media, incubating for a minimum of 14 days and then checking for evidence of microbial contamination. To optimize aseptic transfer of samples into the test media, all testing is performed in a state-of-the-art ISO Class 5 cleanroom.
Samples may be tested using either a direct transfer or membrane filtration method using a soybean casein digest broth (SCDB) and fluid thioglycollate media (FTM). Samples tested by direct transfer are aseptically immersed in the test media. Samples tested by membrane filtration are passed through a 0.45µm filter, and the filter is immersed in the test media. SCDB is used for aerobic and fungal growth at incubation temperatures of 20 – 25°C and FTM is used for anaerobic growth at an incubation temperature of 30 – 35°C.
The successful completion of a USP sterility test does not assure that all products in a sterilization lot are sterile. This is accomplished primarily by validation of the sterilization process or of the aseptic processing procedure which, along with the use of routine controls and monitoring, forms the foundation of a sterility assurance program.
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