There are several methods that can be applied to detect pyrogens: the bacterial endotoxins test (BET – including kinetic chromogenic, kinetic turbidimetric, and gel-clot techniques) historically known as the Limulus amebocyte lysate (LAL) test, the rabbit pyrogen test (RPT), the monocyte activation test (MAT), the recombinant Factor C (rFC) assay, etc.
To select the appropriate test or assay, it is important to understand the difference between pyrogens and endotoxins and what substance each method detects. Pyrogens are a group of substances that elicit a fever, and endotoxins are a member of that group. Thus, all methods that detect pyrogens will also detect endotoxins, but endotoxin-specific tests will not detect the presence of non-endotoxin pyrogens. However, because endotoxins are the most potent, remain toxic after sterilization, and the most difficult of all pyrogens to remove after product contamination, they are often considered the most significant pyrogen. Therefore, the focus often lies only on the detection of endotoxins.
Even though endotoxins are considered to be the most significant, this does not mean that all other pyrogens can be ignored by default. For devices and drug materials, manufacturers should assess the risk of the presence of non-endotoxin pyrogens. If the risk assessment indicates that non-endotoxin pyrogens may be present, it may be more appropriate to use the RPT* or the MAT. This might imply that initial pyrogen (MAT or RPT) testing may be required to demonstrate the absence of non-endotoxin pyrogens before relying solely on BET. In practice this is often resolved by testing three batches to demonstrate that non-endotoxin pyrogens are not present.
All BETs are performed using a lysate from the amebocytes of the blood of the horseshoe crab (Limulus polyphemus), hence, Limulus amebocyte lysate or LAL. This lysate contains an enzyme cascade, beginning with Factor C, which is activated by endotoxins, resulting in a clot (gel-clot technique), change in turbidity (turbidimetric technique), or a change in color (chromogenic technique). The gel-clot technique was the first BET method developed. It renders only qualitative results and is the least sensitive method with a sensitivity ranging from 0.06 EU/mL to 0.015 EU/mL. Method development or validation testing typically requires additional time in comparison to other techniques and in some cases additional product to complete. Due to the additional testing required to validate the method, the testing can also be more expensive to complete. Although the method has some drawbacks, the assay is not impacted by color or particulates and can be helpful for dark colored solutions or products containing particulates that cannot be removed before testing. The gel clot technique is typically considered inferior to the newer, more sensitive, quantitative kinetic chromogenic and turbidimetric techniques. The kinetic techniques have assay sensitivities of 0.005 EU/mL and in specific cases up to 0.001 EU/mL. As the kinetic chromogenic technique measures a change in color, specifically yellow, products which result in a yellow-colored testing solution may not be appropriate for testing using the chromogenic technique. Similarly, as the kinetic turbidimetric technique measures a change in turbidity or cloudiness, products which result in a turbid solution may not be appropriate for testing using the turbidimetric technique.
The rFC assay has a sensitivity of 0.005 EU/mL and is performed using a synthetic reagent which contains a recombinant form of Factor C that has been constructed in vitro. The assay is not susceptible to false positives due to beta-glucans, which come from cellulose and other plant-based products, as the BETs are. As the rFC is synthetic, use of the rFC assay may result in a more sustainable testing plan while also being more environmentally friendly by helping reduce the need for the horseshoe crab blood.
To conclude, the answer to the question of which method to choose is dependent on what you are trying to achieve. If you require a test to detect all pyrogens, the MAT or RPT test are appropriate. If you are required to only test for endotoxins, the choice between the BET an rFC may depend on your sustainability desires or needs while the specific BET technique you choose may be determined based on your desired assay sensitivity, budget, and the specific characteristics of your product.
*(FDA: Guidance for Industry: Pyrogen and Endotoxins Testing: Questions and Answers (2012)
