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{{Short description|Removal of pyrogens from
{{more footnotes|date=May 2012}} <!-- Image with unknown copyright status removed: [[Image:Endo1.jpeg|right]] -->
'''Depyrogenation''' refers to the removal of
A [[wikt:pyrogen|pyrogen]] is defined as any substance that can cause a fever. Bacterial pyrogens include [[endotoxins]] and [[exotoxins]], although many pyrogens are endogenous to the host. Endotoxins include [[lipopolysaccharide]] (LPS) molecules found as part of the cell wall of [[Gram-negative]] bacteria, and are released upon bacterial cell [[lysis]]. Endotoxins may become pyrogenic when released into the bloodstream or other tissue where they are not usually found. Although the colon contains Gram-negative bacteria in abundance, they do not cause a pyrogenic effect as the bacteria are not undergoing gross lysis, and the immune system is not exposed to free endotoxin while the colonic wall is intact.
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=== LAL test ===
{{main|Limulus Amebocyte Lysate}}
Currently, one common method for endotoxin detection is the [[Limulus Amebocyte Lysate]] (LAL) test. This test is based on Dr. Frederik Bang's observation that horseshoe crab blood forms clots when exposed to endotoxins.<ref>{{cite web |last1=Greer |first1=Spencer |last2=Health |first2=JH Bloomberg School of Public |title=Frederik Bang |url=https://www.jhsph.edu/about/history/heroes-of-public-health/frederik-bang.html |website=Johns Hopkins Bloomberg School of Public Health
Since 2003, a synthetic substitute for the LAL test has been commercially available. This recombinant factor C (rFC) test is based on [[Limulus clotting factor C]], the LPS-sensitive part of LAL. The adoption of this test was slow, which began to change in 2016 when the [[European Pharmacopoeia
=== Monocyte Activation Test ===
The [[Monocyte Activation Test]] (MAT) uses the [[monocyte]]s in human blood ''[[in vitro]]'' to detect pyrogens. It was added to the
== 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.<ref>{{cite journal |last1=Sandle |first1=Tim |title=A Practical Approach to Depyrogenation Studies Using Bacterial Endotoxin |journal=Journal of GXP Compliance |date=October 2011 |volume=15 |issue=4 |url=https://www.researchgate.net/publication/282704534}}</ref>
{{main|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.
: Example of using [[anion exchange chromatography]] to purify albumin:<ref>''Chromatographic Removal of Endotoxins and/or Ethanol from Albumin. Application Note 206.'' Pharmacia Biotech., Uppsala, 1990.</ref>
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:* 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 β-interferon. (Dembinski, et al.)<ref>Dembinski, W.; O'Malley, J.A.; Sulkowski, E. Large Scale Purification Procedure for Human Fibroblast Interferon. ''Interferon Scientific Memoranda'', Jan./Feb., 6 (1983).</ref>
{{main|Ultrafiltration}}
:Because the molecular weight of endotoxins is usually over 10 kD, ultrafiltration 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. Commercially available ultra filters have been shown to remove pyrogens to a level below 0.001 EU/ml.{{citation needed|date=November 2010}}
{{main|Distillation}}
: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.
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Because pyrogens are often difficult to remove, inactivation or destruction of the LPS molecule can sometimes be preferable.
{{main|Hydrolysis}}
<!-- Image with unknown copyright status removed: [[Image:Endo3.jpeg|right]] -->
{{main|Oxidation}}
;Heating▼
Heating methods are often used to ensure that glass and other lab equipment are free of pyrogenic material. Heat is applied by baking in a dry heat oven that is designed specifically for the depyrogenation process. Although endotoxins are relatively thermally stable, sufficient heating (250 °C for 30 min) results in a [[Log reduction|3-log reduction]] of endotoxin levels. Due to the high temperature levels, this method is also not suitable when purifying proteins.
: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.▼
===Alkalies===
{{main|Alkali}}
▲
== Preventive methods ==
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== References ==
{{Reflist}}
* 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}}▼
* Tours, N. and Sandle, T. Comparison of dry-heat depyrogenation using three different types of Gram-negative bacterial endotoxin, European Journal of Parenteral and Pharmaceutical Sciences, Volume 13, No.1, 2008,
==External links==
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*[http://www.acciusa.com/bet/update/index.html LAL Update]
*[http://www.acciusa.com/pdfs/newsletter/LAL_Vol.11No.5.pdf.html Depyrogenation LAL Update]
*
▲* 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}}
▲* [https://www.fda.gov/ICECI/Inspections/InspectionGuides/InspectionTechnicalGuides/ucm072918.htm FDA Office of Regulatory Affairs: Inspection Technical Guide, Bacterial Endotoxins/Pyrogens]
▲* [http://textbookofbacteriology.net/endotoxin.html Textbook of Bacteriology]
*[https://www.researchgate.net/publication/282704534_A_Practical_Approach_to_Depyrogenation_Studies_Using_Bacterial_Endotoxin A Practical Approach to Depyrogenation Studies Using Bacterial Endotoxin]
▲* [http://www.horseshoecrab.org/med/med.html Horseshoe Crab Medical Uses]
▲* Tours, N. and Sandle, T. Comparison of dry-heat depyrogenation using three different types of Gram-negative bacterial endotoxin, European Journal of Parenteral and Pharmaceutical Sciences, Volume 13, No.1, 2008, pp17–20
[[Category:Toxicology]]
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