Kerri Hirst, Senior Microbiologist
Sterility testing by USP <71> is a test used in tandem with other sterility assurance procedures and tests to ensure that a product is free of microbial contaminants and safe for patients to use. For a sterility test to perform appropriately, a method suitability validation must be completed on each specific formulation to determine the appropriate test method.
Sterility method suitability testing is performed to determine whether any inhibitory or antimicrobial properties in a drug product will prevent the sterility test from detecting the presence of viable microorganisms. It shows that the sterility test method is valid for the specific drug product and reduces the possibility of a sterile result on a product that is not sterile. Inhibitory properties can vary between drug products and components of a drug product formulation. Active ingredients, inactive ingredients, preservatives and vehicles can all produce inhibitory properties. This highlights the importance that method suitability testing be performed when any change is made to a formulation. Method suitability is formulation specific!
A sterility test method is selected based on the known inhibitory nature of a formulation based on a detailed evaluation of the formulation sheet, including applicable sub-formulations. For a closed membrane filtration method, a volume which is greater than or equal to the volume to be used during the sterility test is passed through two closed system filtration devices. Viable microorganisms will be retained on the filters while the drug product is transferred to a waste container.
The filters are rinsed using fluid A, D or K to remove any lingering drug product from the filters and containers. Fluid thioglycollate medium (FTG) and tryptic soy broth (TSB) are filled independently into each container. Membrane filtration is the preferred method for the sterility test by USP <71> because it separates the product from the potential microbial contaminants which reduces or eliminates the inhibitory properties of the drug. Another test method, direct inoculation, where the product is diluted directly into FTG and TSB to overcome the inhibition of the product, is used when the product to be tested is unfilterable. Certain oils used for vehicles or products that are suspension mixtures can make filtration impossible. The chosen test method is performed using the drug product in 3 TSB and 3 FTG containers, where six test organisms are inoculated into the applicable media, incubated for up to 5 days, and observed for growth.
The sterility method suitability test organisms are a range of different types of organisms that require different types of growth conditions and are representative of common organisms that may be found in the compounding environment.
| Organism | Type | Properties | Oxygen? | Media |
|---|---|---|---|---|
| Staphylococcus aureus | Bacteria | Gram-positive cocci (Spherical) | Aerobic | FTG |
| Bacillus subtilis | Bacteria | Gram-positive Rod, Spore forming | Aerobic | TSB |
| Pseudomonas aeruginosa | Bacteria | Gram-negative Rod | Aerobic | FTG |
| Clostridium sporogenes | Bacteria | Gram-positive Rod, Spore forming | Anaerobic | FTG |
| Candida albicans | Fungi | Budding yeast | Aerobic | TSB |
| Aspergillus brasiliensis | Fungi | Spore forming filamentous mold | Aerobic | TSB |
If all of the organisms grow in the same manner as the positive control and no growth is observed in the negative control, the method suitability has passed and the method may be used for the sterility test. If all the test organisms do not grow, the test must be repeated using a new method that utilizes additional neutralizing practices. Increasing the ability of a test method to neutralize the antimicrobial properties can be done in several ways. For overcoming the inhibition of more antimicrobial products such as antibiotics using closed membrane filtration, a pre-filtration dilution into fluid A can be added. Then, after the diluted sample is passed through the filter, a secondary rinse can be performed. For direct inoculation methods, increasing the dilution or adding USP-approved neutralizers to the media to overcome the inhibitory properties of the product can be done. The appropriate sterility test method can be determined by trying one or more attempts using increasingly more conservative test methods until all six organisms are recovered.
The method determined to be suitable for a specific formulation directly impacts the confidence in all future sterility tests for that product. The formulation must stay the same for the method to be valid for the sterility test on future lots. Changes to any of the components of the formulation or sub-formulation(s) can impact the validity of the sterility test, and method suitability must be repeated. The sterility test is a critical part of the sterility assurance level required by compounders, but it is not the only indicator of sterility assurance. It is up to the compounding facility to develop a sterility assurance plan which should include the sterility test and it is up to ARL Bio Pharma to provide the best sterility test service to meet the need for our clients.
For more information, contact ARL at 800-393-1595 or info@arlok.com.