Endotoxin and Pyrogen Testing PDF

Endotoxin and pyrogen testing   A pyrogen can be defined as a fever producing substance.  Although there were many early reports of fever-producing effects of some substance when injected it was not until late in the nineteenth and early in the twentieth centuries that an understanding of the mic...

Endotoxin and pyrogen testing   A pyrogen can be defined as a fever producing substance.  Although there were many early reports of fever-producing effects of some substance when injected it was not until late in the nineteenth and early in the twentieth centuries that an understanding of the microbiological origin of pyrogens began to emerge. Their true nature and origin were eventually established in 1923 by Siebert who showed conclusively that the pyrogenic effect, resulting from injecting aqueous solutions, was due to the presence of filterable, thermostable substances of bacterial origin, mainly Gram-negative organisms. Pyrogens are now considered to be identical with or very closely related to bacterial endotoxins. Such toxins are now used as standard reference pyrogens.  Certain Gram-positive organisms, fungi and viruses are also capable of eliciting a pyrogenic response. The modern view of the pyrogenic reaction is that the endogenous pyrogen, cytokine, is produced by host cells in response to exposure to exogenous pyrogens, such as endotoxin. The endogenous pyrogen then reaches the hypothalamus where it stimulates heat conservation. A pyrogen test was first introduced into the USP in 1942 and the British Pharmacopoeia in 1944. It is based on observing the effect, if any, on the rectal temperature of rabbits when a sample of the test product is injected slowly into the marginal vein of the ear. Use of the rabbit test has declined in recent years as it has been mostly replaced by the in- vitro Limulus Amoebocyte Lysate (LAL) test where appropriate. The LAL test relies on the formation of a gel from the lysis product of amoebocytes of the giant horseshoe crab (Limulus polyphemus) in the presence of endotoxin in sample. A newer test method is the Monocye Activation Test (MAT). New submissions in Europe will now require the MAT to be performed, when required in lieu of the rabbit test. The Ph. Eur. now states wherever possible and after product -specific validation, the pyrogen test is replaced by the monocyte- activation test (MAT). In June 2021, the European Commission decided to aim towards elimination of the Rabbit Pyrogen test within approximately 5 years. The test should be replaced with a suitable in vitro test, the best option was suggested to be the MAT test ([[https://www.edqm.eu/en/news/european-pharmacopoeia-put-end-rabbit-pyrogen-test]](https://www.edqm.eu/en/news/european-pharmacopoeia-put-end-rabbit-pyrogen-test); accessed Oct 2021).   A revised Ph. Eur. chapter on the Monocyte Activation Test (MAT) came into effect in July 2017. The aim of the test is to facilitate the replacement of pyrogen tests in rabbits. There is also concern regarding the long term use of the horseshoe crab. All new European applications that require non- endotoxin pyrogen testing, must include MAT. The MAT test is also included in the 2012 FDA Guidance. The test is based on being able to quantify a response from the effect on monocytes by pyrogens on releasing cytokines, for example Interleukin -6 (IL-6) or Interleukin -- Beta ( IL-1β). This response can then be measured by for example ELISA The monocytes are obtained in a number of different ways - - - In order to ensure the validity of testing, it shall be confirmed that there are no interfering factors. If dilution is required, ensure dilution steps do not exceed MVD. Add the endotoxin standard to the product dilution at justified concentration of one of the following three methods. In Method A, the added endotoxin level is at or near the middle of the endotoxin standard curve. In Method B, the added endotoxin level is twice the limit of detection. In Method C, the highest concentration ( lowest dilution) that stimulates  the greatest release of the chosen read out, and the 2- fold dilutions immediately below and above the chosen dilution. Preparatory testing may require validation using non- endotoxin pyrogens, depending on the product. Standard curves are required for testing; there should be at least 4 endotoxin concentrations in the required range and at least 4 replicates of each concentration. The table below compares the 3 test methodologies +-------------+-------------+-------------+-------------+-------------+ |   |   | **Rabbit | **Endotoxin | **MAT** | | | | Pyrogen | ** | | | | | Test** | | | | | | | **LAL** | | +=============+=============+=============+=============+=============+ | **Detectabl | Gram | \+ | \+ | \+ | | e | Negative | | | | | pyrogens** | | | | | +-------------+-------------+-------------+-------------+-------------+ | | Gram | \+ | \- | \+ | | | Positive | | | | +-------------+-------------+-------------+-------------+-------------+ | | Fungi | \+ | \- | \+ | +-------------+-------------+-------------+-------------+-------------+ | | Virus | +/-^ 1^ | \- | \+ | +-------------+-------------+-------------+-------------+-------------+ | **Applicati | Pharmaceuti | \+ | \+ | \+ | | ons** | cals | | | | +-------------+-------------+-------------+-------------+-------------+ | | Biologicals | \+ | +/-^ 2^ | \+ | | | e.g. | | | | | | recombinant | | | | | | therapeutic | | | | | | proteins | | | | +-------------+-------------+-------------+-------------+-------------+ | | Blood | \- | \- | \+ | | | products | | | | +-------------+-------------+-------------+-------------+-------------+ | | Cellular | \- | +/- | \+ | | | products | | | | | | e.g. | | | | | | Monoclonal | | | | | | antibodies | | | | +-------------+-------------+-------------+-------------+-------------+ | | Medical | \+ | +/-^3^ | \+ | | | devices | | | | +-------------+-------------+-------------+-------------+-------------+