Depyrogenation
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[edit] Definition
Depyrogenation is the removal of pyrogens from solution. A pyrogen is defined as any substance that can cause a fever. However, this 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 part of the pyrogen's pathogenic effect. A pyrogen can either be an endotoxin or an exotoxin, although most pyrogens are are endogenous. Endotoxins are lipopolysaccharide (LPS) molecules found in the cell wall of gram-negative bacteria, 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.
[edit] Limits
Because endotoxin molecular weight can vary a great deal (10,000 to 1,000,000 Da), endotoxin levels are measured in "endotoxin units" (EU). 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 dosages of endotoxins in the blood stream are often fatal.
The FDA has set the following maximum endotoxin levels in the United States:
- Drug (injectable, non-intrathecal) - 5 EU/kg product
- Drug (injectable, intrathecal) - 0.2 EU/kg product
- Sterile water - 0.25-0.5 EU/ml (depends on intended use)
[edit] Detection
Early endotoxin detection was accomplished by injecting rabbits with the sample and observing their response. Rabbits have similar endotoxin tolerance to humans, and were thus an ideal choice. However, this method was costly, time consuming, and prompted protests from animals rights advocates. But perhaps its biggest drawback of this test was its inability to quantify the endotoxin level.
Currently, the method of choice for endotoxin detection is the Limulus Amebocyte Lysate (LAL) test. This test is based on the observation that horseshoe crab blood forms clots when exposed to endotoxins. The FDA has approved four variations of the LAL test: gel-clot, turbidimetric, colorimetric, and chromogenic assay. This test is fast (approx. 30 minutes) and highly sensitive (up to 0.005 EU/ml sensitivity). However, because it only detects 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.
[edit] Removal
Ion exchange chromatography can be used effectively to remove pyrogens from a sample solution. 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 albumin. Ligands known to bind to endotoxins can be coupled to an anion exchange system to increase 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 polymixin B. However, polymixin B is known to induce production interleukin-1 (an endogenous pyrogen), and thus must be shown to be absent in the final product if used.
Alternative methods for pyrogen removal include the use of cation exchange chromatography, in which the target binds to the column instead of the endotoxin, as is the case in anion exchange chromatography discussed above.
Also, because the molecular weight of endoxins is usually high, ultrafiltration can sometimes be used to perform as a size based separation. However, this can often prove difficult due to the high variability of endotoxin size, and is best used only when all endotoxins present are larger than 300,000 Da.
Distillation is the method of choice for the purification of water.
[edit] Inactivation/Destruction
Because pyrogens are often difficult to remove, inactivation or destruction of the LPS molecule can sometimes be preferable. Acid-base hydrolysis has been shown to cleave the lipid from the polysaccharide in the LPS molecule. The lipid alone is not soluble in water, and is thus unable to bind to endothelial cells, which renders it inactive. However, acid-base hydrolysis can denature a target protein, and is thus unsuitable when purifying a protein.
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.
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 (250°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.
When purifying proteins, sodium hydroxide can be used safely and effectively. In fact, when using an anion exchanger to remove pyrogens, it is necessary to clean the column with NaOH after each batch.
[edit] Preventative Methods
Because virtually all raw materials involved in a production process, including factory employees, can be potential sources of pyrogen contamination, preventative methods can often go a long way to ensuring the final product is free of pyrogens and does not require costly removal or inactivation methods. Filtering chemicals and buffers, applying appropriate hygienic practices, and performing regular tests can all be helpful.
[edit] See Also
[edit] References
- Sofer, G.; Hagel, L. (1997). Handbook of Process Chromatography: A guide to Optimization, Scale-up, and Validation. Academic Press, 158-161. ISBN 0-12-654266-X
- FDA Office of Regulatory Affairs
- Textbook of Bacteriology
