BACTERIAL ENDOTOXIN TESTING
• High sensitivity allows for greater dilution to over- come interference Gel-Clot BET Testing Method Gel-clot Method The formation of a gel-clot indicates the presence of endotoxin in a sample. The method is performed in small test tubes and is read manually by inverting the test tubes. • Requires non-circulating water bath or dry bath incubator
• Manually read test
or potential interference. It is typically performed by testing a series of dilutions of sample with and with- out a Positive Product Control (PPC). PPCs consist of sample with a known amount of endotoxin stan- dard. The purpose is to indicate that added endo- toxin is appropriately detected and that the sample does not interfere with the detection of endotoxin. From the results of the Preliminary Testing, a product dilution and possibly product treatment is selected for the Test for Interfering Factors (see below). The endotoxin limit for the product must be detectable at the dilution selected. Test for Interfering Factors (Validation) The Test for Interfering Factors is performed to validate the test conditions and dilution for the particular sample type. It is accomplished by demon- strating that endotoxin added to the sample in PPCs can be readily detected within required limits. Routine Testing Routine testing is conducted using the sample method preparation and conditions for the Test for Interfering Factors and includes a parallel PPC to check for interference. Tests also include negative controls and appropriate standards. Multiple number of units per lot of finished product should be tested, usually sampled from the beginning, the middle and the end of the production run. For medical devices, aqueous extracts of up to ten units are tested, usually after pooling.
• Reagents of differing sensitivity are available: 0.25, 0.125, 0.06 and 0.03 EU/mL
• May be less sensitive to interference than other methods
• Is the referee method as per BET chapters in the United States, European and Japanese Pharmacopeia Overview of Testing Procedures The following section summarizes the procedures/steps to be taken to perform routine product release testing of a sample in a regulated environment. In an unregu- lated environment, or when testing for informational purposes only, follow the procedures described under Preliminary Testing.
Qualification of Reagent, Technician and Laboratory
The reagent must be tested to ensure that it is per- forming to specification. Technicians must be qualified to perform the test and the absence of significant day to day or inter-technician variability in the laboratory should be documented. This requires testing using endotoxin standards only, not samples. Preliminary Testing Preliminary Testing is not a regulatory requirement, but is an important step to develop a set of conditions for the test method that can be used in the Test for Interfering Factors to demonstrate the absence of in- terference. During Preliminary Testing samples should be characterized for endotoxin contamination and/
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