Introduction to pyrogens and bacterial endotoxin

For those less familiar with pyrogens - and the effects of pyrogenicity - and more specifically, bacterial endotoxin - this week's article takes a look into this important topic, particularly in the context of sterile medicinal products.

Pyrogens

The term pyrogen is derived from the Greek word pyrexia. Pyrogens can be either internal (endogenous) or external (exogenous) to the body. All endogenous pyrogens are cytokines, molecules that are a part of the innate immune system (for example, interleukin 1 (α and β) and interleukin 6 (IL-6)). Exogenous pyrogens can enter the bloodstream via pharmaceutical preparations that are injected into the bloodstream (parenteral products). The most common type is bacterial endotoxin.

Although a 'pyrogen' is introduced into the body as an exogenous agent, its presence causes the release of endogenous factors, that is, the immunological response within the body is the same. This effect includes fever: having a temperature above the normal range due to an increase in the body's temperature set-point.

In the early days of the pharmacopeiae drug substances were classed as apyrogenic or pyrogenic, based, from 1942 and until the 1980s, solely on the ‘pyrogen test’ (whereby a quantity of the drug was injected into three rabbits and the temperature response of the rabbits was noted). The rabbit pyrogen test was first described by Florence Seibert in 1925. It remained the mainstay pyrogen test until the 1980s.

The rabbit test is generally no longer used and it has largely been replaced, for the testing of parenteral drug products, by the LAL (Limulus amoebocyte lysate) test and its recombinant variants (or, in some cases, the Monocyte Activation Test).

LAL and the withering of rabbit testing

The LAL test is a method of the Bacterial Endotoxin Test (BET) for detecting the presence, and to go some way to determining the level, of Gram-negative bacterial endotoxins in a given sample or substance. Current editions of the pharmacopoeia carry statements to the effect that where the term apyrogenic or pyrogen-free is used it should be interpreted as meaning that samples of the product will comply with a limit for bacterial endotoxin.

Gram-negative bacteria

It was not until the early 20th century that an understanding began to emerge in which bacteria could be classified into pyrogenic and non-pyrogenic types, correlatable to their Gram-stain.? Gram staining is a method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative). It is based on the chemical and physical properties of their cell walls. Primarily, it detects peptidoglycan, which is present in a thick layer in Gram-positive bacteria. A Gram-positive results in a purple/blue color while a Gram-negative results in a pink/red color.

Gram-negative bacteria were found to be pyrogenic, Gram-positive bacteria were generally not: and killed cultures of Gram-negative bacteria were comparable to live cultures in their ability to induce fevers.? It was found that the injection of living or killed Gram-negative cells into experimental animals causes a wide spectrum of nonspecific pathophysiological reactions, such as fever, changes in white blood cell counts, disseminated intravascular coagulation, hypotension, shock, and death.

Thus, by the 1920’s it was apparent that sterility in parenteral pharmaceuticals could be no guarantee of non-pyrogenicity, and that if pyrogenicity was to be avoided it was imperative to avoid bacterial contamination at every stage of the manufacture of parenteral pharmaceuticals.?

In recognition that the causative agent of pyrogenicity was filterable and heat stable, efforts were applied to identifying its chemical composition.? Trichloroacetic acid and phenol-water extractions of bacteria were found to be effective in isolating the pyrogenic element from bacteria.? These extracts were chemically identifiable as lipopolysaccharide (or what is commonly described as bacterial endotoxin).

Toxins

Bacteria generate toxins. These can be classified as either exotoxins or endotoxins. Exotoxins are generated and actively secreted; endotoxins remain part of the bacteria. Usually, an endotoxin is part of the bacterial outer membrane, and it is not released until the bacterium is killed by the immune system.

Endotoxin refers to a component of the outer cell membrane of Gram-negative bacteria, which is known to induce a febrile response in humans and other mammals. The endotoxin complex contains many cell wall components including, but not limited to, phospholipids, lipoproteins, and lipopolysaccharides. Lipopolysaccharide (LPS) is the biologically active portion of both naturally occurring and laboratory-prepared endotoxin complexes.

Exotoxins are highly potent and can cause major damage to the host. An example is botulinum toxin produced by Clostridium botulinum. They are not a concern in this unit because the toxic properties of most exotoxins can be inactivated by heat or chemical treatment.

The structural rigidity of the bacterial cell wall is conferred by a material called peptidoglycan (also known as murein). It is a polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of bacteria, forming the cell wall.?

Form and structure

In Gram-positive bacteria peptidoglycan is present as a thick layer which is outer-most in the cell wall. In Gram-negative bacteria, the peptidoglcan is only a thin layer and it is not the outermost layer.? Gram-negative bacteria are sometimes described as having a cell envelope rather than a cell wall.? The term envelope better describes the loosely attached layer of material called lipopolysaccharide which is located outside a thin structural layer of peptidoglycan.

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The outer layer of this lipopolysaccharide envelope is a permeability barrier effective against the diffusion of exo-enzymes into the external environment.? This is an evolutionary feature that has arisen to allow Gram-negative bacteria to survive and increase in numbers in environments such as water in which there are only low concentrations of organic nutrients.? Lipopolysacccharide is pyrogenic.? Bacterial endotoxin is a synonym for lipopolysaccharide. Although intimately associated with the cell envelope of Gram-negative bacteria, lipopolysaccharide is constantly shed by the bacteria into the environment, much like the shedding of the outer layers of human skin.? When Gram-negative bacteria die and lyse, all of their lipopolysaccharide is shed into the environment.

Furthermore, when bacterial cells are lysed by the immune system, fragments of membrane containing lipid A are released into the circulation, causing fever, diarrhea, and possible fatal endotoxic shock (also called septic shock) (12). There are some other substances which are also pyrogenic.? They are unusual and are extremely rarely found associated with pharmaceutical preparations.

Immune reaction

With the immune response: once inside the body, natural defense cells like macrophages and monocytes recognize the bacteria as foreign. This recognition process is mediated by the antigens in bacteria which include the O-antigen of LPS. The bacteria may sometimes evade destruction but are normally degraded by the body’s defense cells. The endotoxins located within the bacteria are then released into the circulation and exert their deleterious effects. The defense cells of the body also release substances that may stimulate pathways that compound the negative effects of endotoxins.

The complement cascade, through substances C3a and C5a, causes the release of histamine, a major effector for the allergic response. The complement cascade generally causes vasodilation and inflammation.

The inflammatory response is mediated through the release of substances which include the cytokines interleukins, prostaglandins, and tumor necrosis factor. Interleukins are pyrogenic, they cause the febrile response. Aside from inflammation, these substances also mediate the shock response.

The coagulation pathway is also activated. There is generally aggregation of platelets, dilatation of the blood vessels, and increased leakage of the vessel walls. The substrates required to form a blood clot may be diminished or depleted in endotoxemia. This depletion, along with vessel dilatation and increased leakage contributes to the occurrence of hemorrhages in bacterial infection.

Lipopolysaccharide has three distinct chemical regions, as illustrated below:

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• An inner core called Lipid A
• An intermediate polysaccharide layer
• An outer polysaccharide side chain.?

Lipid A is a powerful biological response modifier that can stimulate the mammalian immune system. Thus lipid A, embedded in the bacterial outer membrane, is responsible for pyrogenicity.

Endotoxin presents a risk to pharmaceutical processing due to its primary source: water and due to the fact that it is filterable and is unaffected by steam sterilization. Thus the control of water is of great importance.

Endotoxin risks

Although all bacteria have some associated endotoxin, the most potent source of endotoxin is from Gram-negative bacteria.? Endotoxins may be shed from viable bacteria during growth, or they may be associated with non-viable bacteria.? Importantly, endotoxicity is not a function of microbial viability (that is, microbial cells may be dead but endotoxin molecules can continue to be present.)? The most common habitat for Gram-negative bacteria is water where they have evolved to be able to survive and increase in numbers with minimal nutritional support and to be able to adapt their metabolism to metabolise complex organic macromolecules that other bacteria cannot.?

Water

In any form of pharmaceutical manufacture water is one of the most serious potential sources of microbiological contamination.? Water cannot be totally excluded from sterile products manufacturing facilities.? The primary focus of endotoxin control in pharmaceutical manufacture is on controlling it at its source – water.? If endotoxin is not controlled at its source it has the potential to create difficulties through manufacture to the finished product, potentially leaving no recourse but rejection.? Endotoxin is practically impossible to remove terminally from pharmaceutical dosage forms.

Dr. Tim Sandle is a pharmaceutical microbiologist and the manager of the popular Pharmaceutical Microbiology Resources website.

Further reading

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• Guy, D. (2003): “Endotoxins and Depyrogenation” in Hodges, N. and Hanlon, G. (Eds.). Industrial Pharmaceutical Microbiology: Standards and Controls, Euromed, pp. 12.1 –12.15
• Cooper, J.F. and Williams, K. (2007) Pyrogenicity Case Studies,? in Williams, K.(Ed).? Endotoxins: Pyrogens, LAL Testing And Depyrogenation, New York: Infroma Healthcare
• Sandle, T. (2004): ‘Three Aspects of LAL Testing: Glucans, Depyropgenation and Water System Qualification’, PharMIG News No. 16, June / July 2004, pp3-12
• McCullogh, K.C. and Weider-Loeven, C. (1992): ‘Variability in the LAL Test: Comparison of Three Kinetic Methods for the Testing of Pharmaceutical Products’, Journal of Parenteral Science and Technology, 46 93): 69-72
• Brandberg, K., Seydel, U., Schromm, A.B., Loppnow, H., Koch, M.H.J., and Rietschel, E. T. (1996): ‘Conformation of Lipid A, the Endotoxic Center of Bacterial Lipopolysaccharide’, Journal of Endotoxin Research, 3(3): 173-178