Depyrogenation refers to the removal of pyrogens from solution, most commonly from injectable pharmaceuticals.
A pyrogen is defined as any substance that can cause a fever. A pyrogen can either be an endotoxin or an exotoxin, although most pyrogens are endogenous. Endotoxins (not to be confused with Enterotoxin) are potentially toxic, natural compounds found inside Pathogens such as Bacteria. An exotoxin is a soluble Protein excreted by a Microrganism, including bacteria, Fungi, Algae, and Protozoa. 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. Lipopolysaccharides ( LPS) are large Molecules consisting of a Lipid and a Polysaccharide joined by a Covalent bond; they are found Gram-negative bacteria are those Bacteria that do not retain Crystal violet dye in the Gram staining protocol 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.
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. Fever (also known as pyrexia, from the Greek pyretos meaning fire or a febrile response, from the Latin word Febris Rather, the fever is caused as a direct result of pyrogen exposure and is one of the symptoms of septic shock. Septic shock is a very serious medical condition caused by decreased tissue perfusion and oxygen delivery as a result of Infection and Sepsis, though the 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. The endothelium is the thin layer of cells that line the interior surface of Blood vessels forming an interface between circulating Blood in the macrophages and monocytes) and causes them to secrete increased levels of pro-inflammatory cytokines and nitric oxide, which leads to septic shock. Macrophages ( Greek: "big eaters" from makros "large" + phagein "eat" ( Mø) are cells within the tissues that Monocyte is a type of Leukocyte, part of the Human body 's Immune system.
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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. Intrathecal is an adjetive that refers to something that happens inside of the spinal canal 2 EU/kg product
- Drug (injectable, non-intrathecal) - 5 EU/kg product
- Sterile water - 0. 25-0. 5 EU/ml (depends on intended use)
Pyrogen Detection
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 animals rights advocates. But perhaps the biggest drawback of this test was its inability to quantify the endotoxin level.
LAL Test
Currently, the method of choice for endotoxin detection is the Limulus Amebocyte Lysate (LAL) test. Limulus amebocyte lysate (LAL is an aqueous extract of blood cells ( Amoebocytes from the Horseshoe crab, Limulus polyphemus. 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. An ion is an Atom or Molecule which has lost or gained one or more Valence electrons giving it a positive or negative electrical charge Glucans from carbohydrate chromatography matrices can also lead to false positives. A glucan molecule is a Polysaccharide of D- Glucose monomers linked by glycosidic bonds.
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.
Endotoxins are negatively charged, and will bind to an anion exchanger. Ion-exchange chromatography (or ion chromatography) is a process that allows the separation of Ions and Polar molecules based on the charge properties of Ion exchange is an exchange of Ions between two Electrolytes or between an electrolyte Solution and a complex. 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). Albumin (Latin albus white refers generally to any Protein with water Solubility, which is moderately soluble in concentrated salt solutions and experiences 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. In Chemistry, a ligand is either an Atom, Ion, or Molecule (see also Functional group) that bonds to a central metal generally Typical examples of endotoxin binding ligands include histamine, nitrogen-containing heterocyclic compounds, and polymyxin B. Histamine is a Biogenic amine involved in local immune responses as well as regulating physiological function in the gut and acting as a Neurotransmitter Polymyxin B is an antibiotic primarily used for resistant Gram negative infections 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. Polymyxin B is an antibiotic primarily used for resistant Gram negative infections
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. Ion exchange is an exchange of Ions between two Electrolytes or between an electrolyte Solution and a complex. 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 purification. (Dembinski, et al)
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.
This method is also based on the large molecular weight and heat stability of endotoxins. Distillation is a method of separating Mixtures based on differences in their volatilities in a boiling liquid mixture 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.
Inactivation/destruction
Because pyrogens are often difficult to remove, inactivation or destruction of the LPS molecule can sometimes be preferable.
Acid-base hydrolysis This method has been shown to cleave Lipid A from the polysaccharide in the LPS molecule (see right). Hydrolysis is a Chemical reaction during which one or more water molecules are split into hydrogen and hydroxide ions which may go on to participate in further reactions The lipid moiety alone is not soluble in water. Lipids are broadly defined as any fat- Soluble ( lipophilic) naturally-occurring Molecule, such as fats oils waxes cholesterol sterols fat-soluble Thus unable to bind to endothelial cells, it is rendered inactive. The endothelium is the thin layer of cells that line the interior surface of Blood vessels forming an interface between circulating Blood in the 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. Redox (shorthand for reduction-oxidation reaction describes all Chemical reactions in which atoms have their Oxidation number ( Oxidation state 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
Heating methods such as autoclaving are often used to ensure that glass and other lab equipment are free of pyrogenic material. An autoclave is a pressurized device designed to heat aqueous solutions above their Boiling point at normal atmospheric pressure to achieve sterilization 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.
Sodium Hydroxide
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.
Preventative methods
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.
See also
References
- Sofer, G. Endotoxins (not to be confused with Enterotoxin) are potentially toxic, natural compounds found inside Pathogens such as Bacteria. Downstream processing refers to the recovery and purification of biosynthetic products particularly pharmaceuticals from natural sources such as animal or plant tissue or fermentation ; 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
- Horseshoe Crab Medical Uses
- Chromatographic Removal of Endotoxins and/or Ethanol from Albumin. Application Note 206. Pharmacia Biotech. , Uppsala, 1990.
- Dembinski, W. ; O'Malley, J. A. ; Sulkowski, E. Large Scale Purification Procedure for Human Fibroblast Interferon. Interferon Scientific Memoranda, Jan. /Feb. , 6 (1983).
- Biocompare.com
Ref. on Endotoxin binding CD14 in humans: www. monozyten. de