Pyrogens are fever-inducing substances primarily derived from microorganisms such as bacteria, viruses, yeasts, molds, or chemical substances, e.g. primary packaging materials. Pyrogens entering the bloodstream may interact with the host immune system to cause inflammation, fever, chills, shock and, in severe cases, death.
Pyrogens include membrane-derived compounds like lipid-polysaccharides (LPS), flagellin, peptidoglycans, lipids, proteins, etc. They are often heat stable and therefore difficult to remove during manufacturing. The most potent type of pyrogens are bacterial endotoxins which are derived from the cell wall of gram-negative bacteria. Pyrogens that derive from microorganisms other than gram-negative bacterial are collectively referred to as non-endotoxin pyrogens (NEPs).
There are a variety of methods that can be used to detect pyrogens: The traditional qualitative approach has been the rabbit pyrogen test (RPT), which involves measuring the body temperature increase in rabbits following injection of a product potentially containing pyrogens. The monocyte activation test (MAT) was introduced as a sustainable, in vitro alternative to the use of experimental animals. The MAT is based on measuring the release of pro-inflammatory cytokines from cultured human blood monocytes in response to pyrogenic contaminants. MAT kits, such as the PyroCell® MAT System, which measure release of IL-6 are especially useful early in drug development when the presence of all pyrogens should be examined, and testing is not focused specifically on endotoxins.
Bacterial endotoxins are the most potent pyrogenic contaminants and are ubiquitous. Thus, we must try to keep the endotoxin levels as low as possible for all injectable drugs and implantable medical devices. The bacterial endotoxins test (BET) has widely replaced the RPT for pharmaceutical and biotechnology products. Traditional BET using Limulus amebocyte lysate (LAL) tests contain specialized blue blood cells from the wild Atlantic horseshoe crab, Limulus polyphemus, as a component because they react to the presence of endotoxins in a way that can be measured and quantitated. (Similarly, tests available in Asia include cells from an Asian horseshoe crab species, Tachypleus spp., and are called TAL assays.) Sustainable tests, using a recombinant version of the first enzyme in the LAL clotting cascade, such as the PyroGene® rFC Assay, do not rely on the blood of horseshoe crabs. The use of an alternative method reduces the demand on a natural resource and can help meet supply chain sustainability initiatives. They are gaining usage across the globe as companies seek to reduce their reliance on natural resources.