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For a printer-friendly version, click here. To access our Biotech/Biopharm catalog of services, click here. Guidelines to Sterility Testing for Biologics Overview Guidelines for sterility testing of biologics is addressed in the various worldwide pharmacopeias (e.g., USP, EP and JP), Section 21 of the Code of Federal Regulations (CFR), International Conference on Harmonisation (ICH) and Food and Drug Administration Points to Consider (PTC) documents. This document provides a brief summary of the test methods and sample requirements for the most common types of biological products. The outline below represents existing regulations and current industry practice. Exceptions may exist depending on the product and process from which it was generated. Sterility Testing Methods Biological products manufactured under GMP conditions require sterility testing performed under GMP guidelines. There are two common types of sterility test methods: • Immersion (Direct Inoculation) The immersion (direct inoculation) method requires the test article be inoculated directly into test media. • Membrane Filtration The membrane filtration method requires the test article to first pass through a size exclusion membrane capable of retaining microorganisms. The filter is rinsed and then the membrane is transferred into the appropriate test medium. The pharmacopeias and 21 CFR 610.12 recommend using two media for both the immersion and membrane filtration methods. In both test methods the test article or membrane is incubated for 14 days in the test media. The majority of biological samples will be tested using the immersion method. However, for test articles with large volumes in which the entire contents must be tested or if there is a substance within the test article that is known or determined to be bacteriostatic or fungistatic (e.g., cell culture containing methotrexate), the membrane filtration method may be required. AppTec provides sterility testing protocols that comply with both USP and 21 CFR 610.12 guidelines and can be conducted using the immersion or membrane filtration method. Protocols can be customized for those clients needing to meet EP or JP requirements. Sterility Test Validation It is important to determine if the test article that will be tested for sterility contains elements that will interfere with the growth of microorganisms within the growth media used for the assay. [This validation testing is commonly referred to as a Bacteriostasis/Fungistasis (B/F) test.] The validation test is typically required only once for a given sample type, provided that no additional changes to the source, product, formulation or manufacturing process occurred. However, validation testing could be performed on a periodic basis to confirm that no significant changes have occurred to the product or process that may affect the sterility assay results. Sample Requirements by Test Article Type Important note regarding samples for sterility test validation: Sterility test validation (B/F test) can be performed concurrently with the sterility test using an additional matching quantity of identical samples submitted by the client. Or, at the client’s discretion, once the sterility test is completed, the sterility test samples themselves (post-sterility-test samples) can be used for the validation testing so additional samples are not needed. Cell & Virus Bank Testing Cell and virus banks manufactured under cGMP conditions require sterility testing be performed per the appropriate test methods outlined in the pharmacopeias and the 21 CFR 610.12. Neither the USP, EP nor 21 CFR 610.12 provide sampling requirement guidelines for cell and virus banks. However, ICH Q5D “Derivation and Characterization of Cell Substrates Used for the Production of Biotechnological/Biological Products” states that sterility testing will be performed on individual containers. It also states that 1% of the bank or no less than 2 containers will be tested. This recommendation has become a standard industry practice. Unprocessed Bulk Testing (For Protein and Virus Products) Within the biotherapeutics industry “Unprocessed Bulk” has many other descriptions, including cell/viral harvest, clarified cell/viral harvest, end of production cells or cells at the limit of in vitro cell age. Although the pharmacopeias and 21 CFR 610.12 do not reference or provide sterility guidelines for these sample types, the FDA documents “”Points to Consider in the Characterization of Cell lines Used to Produce Biologicals” (1993) and “Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use” (1997) do reference the need for sterility or bioburden testing on each unprocessed bulk lot. These two documents reference the 21 CFR 610.12 for guidelines on the appropriate sterility test methods to use. Since there is no reference in the pharmacopeias or 21 CFR 610.12 specifically related to unprocessed bulk material, no sampling guidelines are available either. However, industry practice is to use the sampling guidelines stated for bulk drug substance as detailed in the 21 CFR 610.12. Thus 10 mL/media (for a total of 20 mL) is recommended for sterility testing of unprocessed bulk material. Bulk Drug Substance (BDS) Testing Sterility testing is required on each manufactured BDS lot. Sampling requirements for Bulk Drug Substance is defined in 21 CFR 610.12, which states that no less than 10 mL will be tested. It is not clear if that is meant for each media or if the amount is to be split into the two test media. Industry practice has taken a conservative approach and 10 mL of BDS is tested in each media. Thus a total of 20 mL is required for sterility testing. Final Drug Product (FDP) Testing Sterility testing is required on each manufactured FDP lot. Sampling requirements for final drug product are provided in the pharmacopeias. The following tables outline the requirements as stated in the current USP. | Batch Size | Minimum Number to be Tested in Each Media | | 100 or less | 10% or 4 containers, whichever is greater | | 101 – 500 | 10 containers | | >500 | 2% or 20 containers, whichever is less | | Bulk – Up to 4 containers | Each container | | 5 – 50 | 20% or 4 containers, whichever is greater | | >50 | 2% or 10, whichever is greater |
Volume / Container | Minimum Quantity to Test in Each Media | | Less than 1 mL | The entire contents of each container | | 1 – 40 mL | Half the contents of each container,
but not less than 1 mL | | 41 – 100 mL | 20 mL | | >100 mL | 10% of the contents of the container,
but not less than 20 mL |
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