By Craig Spitzmiller, Molecular Biologist, NJ Laboratories
Let’s start with the basics! What exactly are Endotoxins and Pyrogens?
Endotoxins, also known as Lipopolysaccharides (LPS), are the component of the outer membrane of gram-negative bacteria and are released into circulation upon disruption of the intact bacteria via death or cell lysis1.
Pyrogens (derived from pyro- meaning “heat” and -gen meaning “that which produces”) are fever-inducing substances usually derived from microorganisms such as endotoxins or lipopolysaccharides (LPS). When present systemically in sufficient quantity, Pyrogens can lead to severe signs of inflammation, shock, organ failure, and even death, in humans2.
Endotoxins are the most ubiquitous form of Pyrogen, and as terms, can usually be used interchangeably3. They are widely present in the environment and can be found in things such as dust, animal waste, foods, and any materials generated from, or exposed to, Gram-negative bacterial products4. In general, humans as products of the natural world, can coexist with microbes in the environment such as endotoxins and other pyrogens without much issue. The body’s natural defenses usually contain these microorganisms to areas where they can be tolerated, such as the skin and the digestive tract. However, the parenteral administration of a pharmaceutical drug, and certain medical devices, allows a pyrogen, if present, to bypass the body’s natural defenses. The host’s response is mediated through the white blood cells, which in turn release their own kind of endogenous pyrogen and initiate a fever producing response and a multitude of other biological reactions to fight off the foreign substance3.
During the advent of injectable pharmaceutical products, there was no method for testing for pyrogenic contaminants. If a patient received an injection, there was a high likelihood that they would spike a fever, which at the time was termed “injection fever”5.
It is for this reason, that it is especially important to ensure that any injectable and transplantable products introduced into the human body are endotoxin-free. Simply performing sterility or plate-count studies is inadequate, as non-viable gram-negative contaminants can still introduce endotoxins into a product and pose a serious health risk. Usual sources of pyrogen contamination in pharmaceutical products are: the water used as the solvent, or in pharmaceutical processing; packaging components; the chemicals, raw materials, or equipment used in the preparation of the finished product6. Good practice would be to monitor and control the microbiological and endotoxin levels at all steps in the manufacturing process, including potential sources for contamination.
Bacterial endotoxins are notoriously difficult to remove from medical devices and finished parenteral products, and procedures such as heating, filtration, or adsorption techniques do not eliminate them from parenteral solutions. In order to reduce the risk for the most vulnerable of patients, such as those in intensive care, infants, elderly, and the immunocompromised, all ingredients must be kept pyrogen free in the first place3,5.
The Bacterial Endotoxins Test (BET) detects and quantifies endotoxins by utilizing Limulus Amoebocyte Lysate (LAL), derived from of all things, the blood of Horseshoe crabs. When LAL comes into contact with bacterial endotoxins, it produces a gelation reaction, which is the basis for BET. The test is simple to perform, and highly sensitive7. New Jersey Laboratories currently offers all three methods of endotoxin detection described in the United States Pharmacopeia, General Chapter <85> “Bacterial Endotoxins Test”. Our highly qualified technicians perform and meticulously document endotoxin testing via Gel-clot, Turbidimetric or Chromogenic methods. The sensitivity of these tests can be adjusted to suit the client’s needs. Detailed results are delivered to our clients so they can rest assured as to the quality of their products, as well as the safety of those who need them most.
1. Maud B. Gorbet, Michael V. Sefton, in The Biomaterials: Silver Jubilee Compendium, 2004
2.P.E. Boucher, in Immunopotentiators in Modern Vaccines (Second Edition), 2017
3.DEPT. OF HEALTH, EDUCATION, AND WELFARE PUBLIC HEALTH SERVICE FOOD AND DRUG ADMINISTRATION *ORA/ORO/DEIO/IB* 1/12/79 Number: 32
“PYROGENS, STILL A DANGER”
4. P.J. Bertics, M.L. Gavala, L.C. Denlinger, Encyclopedia of Respiratory Medicine 2006, Pages 80-85 “ENDOTOXINS”
5. Felicity Thomas “The Importance of Endotoxin Testing” Pharmaceutical Technology’s In the Lab eNewsletter-08-07-2019, Volume 14, Issue 8
6. DEPT. OF HEALTH, EDUCATION, AND WELFARE PUBLIC HEALTH SERVICE FOOD AND DRUG ADMINISTRATION *ORA/ORO/DEIO/IB* 3/20/85 Number: 40
7. Iwanaga S (May 2007). “Biochemical principle of Limulus test for detecting bacterial endotoxins”. Proceedings of the Japan Academy. Series B, Physical and Biological Sciences