Pyrogens as fever-inducing agents can be a major health hazard in parenterally applied drugs. For the control of these contaminants, pyrogen testing for batch release is required by pharmacopoeias. This has been done either by the in vivo rabbit pyrogen test (since 1942) or the limulus amoebocyte lysate test (LAL), since 1976. New approaches include cell-based assays employing in vitro culture of human immune cells which respond e.g. by cytokine production (IL-1Î²; IL-6) upon contact with pyrogens.
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Six variants of these assays have been validated in a collaborative international study. The recent successful development of cryopreservation methods promises to make standardized immunoreactive primary human blood cells available for widespread use. Furthermore, the pretesting of donors for infectious agents such as HIV or hepatitis has made it possible to develop a safe and standardised reagent for pyrogen testing. Using a total of 13 drugs, we have validated the pyrogen test based on fresh and cryopreserved human whole blood in four laboratories. The test reached > 90% sensitivity and specificity. In contrast to the LAL, the test was capable of detecting non-endotoxin pyrogens derived from Gram-positive bacteria or fungi.(1)
Types of pyrogens:
A pyrogen can be an endtoxin or an exotoxin, although most pyrogens are endogenous.
Endotoxins: are lipopolysaccharide (LPS) molecules found as part of the cell wall of gram-negative bacteria (see right), and are released primarily upon cell lysis. Endotoxins: are typically only toxic when found in the bloodstream, and gram-negative bacteria exist routinely in human intestines, but do not cause a pyrogenic effect. (Sofer, G .1997).
It is the removal of pyrogens from drugs, mostly from injectable pharmaceuticals preparations.
The fever inducing aspect of pyrogens does not refer to fever as part of a normal immune response, as may be the case with the flu, for example. Rather, the fever is caused as a direct result of pyrogen exposure and is one of the symptoms of septic shock. When the LPS is released upon cell lysis, “Lipid A” binds to LPS-binding proteins in the bloodstream, which interact with CD14 receptors on endothelial cells (esp. macrophages and monocytes) and causes them to secrete increased levels of pro-inflammatory cytokines and nitric oxide, which leads to septic shock. (Sofer, G.1997).
Maximum acceptable endotoxin levels
Because endotoxin molecular weight can vary a great deal (10,000 to 1,000,000 Da), endotoxin levels are measured in “endotoxin units” (EU). One EU is approximately equivalent to 100 pg of E. Coli lipopolysaccharide — the amount present in around 105 bacteria. Humans can develop symptoms when exposed to as little as 5 EU/kg body weight. These symptoms include, but are not limited to, fever, low blood pressure, increased heart rate, and low urine output; and even small doses of endotoxin in the blood stream are often fatal.
The FDA has set the following maximum permissible endotoxin levels for drugs distributed in the United States:
- Drug (injectable, intrathecal) – 0.2 EU/kg product
- Drug (injectable, non-intrathecal) – 5 EU/kg product
- Sterile water – 0.25-0.5 EU/ml (depends on intended use). (Sofer, G.1997).
Pyrogen detection: by two testes
1) Rabbit Test
Early endotoxin detection was accomplished by injecting rabbits with the sample and observing the response in their body temperature. Rabbits have similar endotoxin tolerance to humans, and were thus an ideal choice. However, this method was costly, time consuming, and prompted protests from animal’s rights advocates. But perhaps the biggest drawback of this test was its inability to quantify the endotoxin level. (Sofer, G.1997).
2) LAL Test
Currently, the method of choice for endotoxin detection is the Limulus Amebocyte Licata (LAL) test. This test is based on Dr. Frederik Bang’s observation that horseshoe crab blood forms clots when exposed to endotoxins. Amoebocyte extract from horseshoe crab blood is mixed with a sample suspected of endotoxin contamination, and a reaction is observed if endotoxins are present. The FDA has approved four variations of the LAL test: gel-clot, turbidimetric, colorimetric, and chromogenic assay. The differences in these variations refer to the characteristics of the amoebocyte/endtoxin reaction (e.g. gel-clot produces a precipitate and colorimetric changes color). This test is fast (approx. 30 minutes) and highly sensitive (up to 0.005 EU/ml sensitivity). However, because it only detects LPS endotoxins, some pyrogenic materials can be missed. Also, certain conditions (sub-optimal pH conditions or unsuitable cation concentration) can lead to false negatives. Glucans from carbohydrate chromatography matrices can also lead to false positives.
Pyrogen removal (Depyrogenation)
Pyrogens can often be difficult to remove from solution due to the high variability of their molecular weight. Pyrogens are also relatively thermally stable and insensitive to pH changes. However, several removal techniques exist. (Hagel, L.1997)
Ion exchange chromatography
Endotoxins are negatively charged, and will bind to an anion exchanger. If the target substance is not also negatively charged, it will pass through the column before the endotoxin, and an effective separation can be achieved. This method is sometimes used in the purification of albumins (details follow). Ligands of known affinity to endotoxins can be coupled to an anion exchange system to increase its endotoxin binding strength and further improve the purity of the final product. Typical examples of endotoxin binding ligands include histamine, nitrogen-containing heterocyclic compounds, and polymyxin B. However, polymyxin B is known to induce production of interleukin-1, an exogenous pyrogen, and thus must be shown to be absent in the final product if used. (Hagel, L.)
Example of using anion exchange chromatography to purify albumin (Uppsala):
2% of the endotoxin does not bind to the column. However, this 2% washes out before the albumin peak, and can thus be removed simply by starting collection after this 2% has washed out.
10% of the endotoxin that does bind to the column (9.8% of the original total) will eventually wash out after the albumin peak. This can be prevented from entering the final product by stopping collection before this happens.
The remaining 90% of the bound endotoxin (88.2% of the original total) must be cleaned off the column using NaOH
An alternative to anion exchange is cation exchange chromatography, in which positively charged solutes bind to the solid chromatographic media. In this method, the target binds to the column instead of the endotoxin. The endotoxin then washes through the column, and a pure target is later eluted off the column. Cation exchange chromatography has been shown to effectively purify Î²-interferon purification. (Hagel, L.1997)
Because the molecular weight of endotoxins is usually over 10 kD, ultra filtration can sometimes be used to perform as a size based separation. Due to the high variability of endotoxin size, it can be difficult to select the correct membrane; hence this method is best used only when all endotoxins present are larger than 300,000 Da. (Hagel, L.1997)
This method is also based on the large molecular weight and heat stability of endotoxins. Low molecular-weight solvents can be easily purified by boiling and collecting the condensed vapor in an endotoxin free vessel (see “heating” below). The large LPS molecules do not easily vaporize, and are thus left behind in the heating vessel. This is the method of choice for the purification of water. (Hagel, L.1997)
Because pyrogens are often difficult to remove, inactivation or destruction of the LPS molecule can sometimes be preferable.
this method has been shown to cleave Lipid A from the polysaccharide in the LPS molecule (see right). The lipid moiety alone is not soluble in water. Thus unable to bind to endothelial cells, it is rendered inactive. However, acid-base hydrolysis can denature a target protein, and is thus unsuitable when purifying a protein. (Dembinski, W.1983)
Oxidation using hydrogen peroxide is often used as a low cost pyrogen destroying solution. The mechanism for this destruction is unknown, but hydrogen peroxide can easily be removed further downstream in the purification process, and is therefore a useful method of pyrogen removal. However, like acid-base hydrolysis, it is not suitable when purifying proteins. (Dembinski, W.1983)
Heating methods such as autoclaving are often used to ensure that glass and other lab equipment are free of pyrogenic material. Although endotoxins are relatively thermally stable, sufficient heating (250Â°C for 30 min) results in a 3log reduction of endotoxin levels. Due to the high temperature levels, this method is also not suitable when purifying proteins. (Dembinski, W.1983)
When purifying proteins, sodium hydroxide (NaOH) can be used safely and effectively. It is also widely used for depyrogenation of non-autoclavable equipment (e.g. plastics) and chromatography columns. In fact, when using an anion exchanger to remove pyrogens, it is necessary to clean the column with NaOH after each batch. (Dembinski, W.1983)
Because virtually all raw materials involved in a production process, including factory employees, can be potential sources of pyrogen contamination, raw material screening and depyrogenation can often go a long way to ensuring the final product is free of pyrogens and does not require costly removal or inactivation methods. Ultrafiltration of chemicals and buffer solutions, applying appropriate hygienic practices, and performing regular tests can all be helpful. (Dembinski, W.1983)
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Infection, fever, and exogenous and endogenous pyrogens
For many years, it was thought that bacterial products caused fever via the intermediate production of a host-derived, fever-producing molecule, called endogenous pyrogen (EP). Bacterial products and other fever-producing substances were termed exogenous pyrogens. It was considered highly unlikely that exogenous pyrogens caused fever by acting directly on the hypothalamic thermoregulatory center since there were countless fever-producing microbial products, mostly large molecules, with no common physical structure. In vivo and in vitro, lipopolysaccharides (LPSs) and other microbial products induced EP, subsequently shown to be interleukin-1 (IL-1). The concept of the `endogenous pyrogen’ cause of fever gained considerable support when pure, recombinant IL-1 produced fever in humans and in animals at subnanomolar concentrations. Subsequently, recombinant tumor necrosis factor- (TNF-), IL-6 and other cytokines were also shown to cause fever and EPs are now termed pyrogenic cytokines. However, the concept was challenged when specific blockade of either IL-1 or TNF activity did not diminish the febrile response to LPS, to other microbial products or to natural infections in animals and in humans. During infection, fever could occur independently of IL-1 or TNF activity. The cytokine-like property of Toll-like receptor (TLR) signal transduction provides an explanation by which any microbial product can cause fever by engaging its exact TLR on the vascular network supplying the thermoregulatory center in the anterior hypothalamus. Since fever induced by IL-1, TNF-, IL-6 or TLR ligands requires cyclooxygenase-2, production of prostaglandin E2 (PGE 2) and activation of hypothalamic PGE2 receptors provides a unifying mechanism for fever by endogenous and exogenous pyrogens. Thus, fever is the result of either cytokine receptor or TLR triggering; in autoimmune diseases, fever is mostly cytokine mediated whereas both cytokine and TLR account for fever during infection. (DinarelloÂ CA.2004)
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