pyrogen test

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pyrogen test ,aimst

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The discovery that aqueous solutions may lead to an increase in body temperature when injected into a patient dates back to the 19th century. The agents responsible for this fever were termed ‘pyrogens’ Pyrogens

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Gram-positive bacteria mycobacteria fungi and also viruses A pyrogen is any substance that, when injected into a mammal, elicits a rise in body temperature

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The most common pyrogens to the pharmaceutical industry, are produced by Gram-negative bacteria and are termed endotoxins. They are lipopolysaccharides (LPS) found in the cell walls.

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The release of LPS from bacteria takes place after death and lysis. Many Gram-negative bacteria, e.g. Escherichia coli and Proteus, Pseudomonas, Enterobacte r and Klebsiella species produce pyrogenic LPS.

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Characteristics of bacterial endotoxin LPS which is composed of two main parts: hydrophilic polysaccharide chain with antigenic regions, hydrophobic lipid group termed lipid A Structure of endotoxins :

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The molecular size of the polysaccharide chain is very variable, and consequently the molecular weight of the LPS may vary from 5000 to 25000 to several million Daltons. The endotoxin (LPS) characteristics Thermostable (withstand 120°C for over 3 hours) water-soluble unaffected by the common bactericides

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non-volatile insensitive to pH changes These are the reasons why pyrogens are difficult to destroy once produced in a product Extremes of pH are required for rapid destruction of the LPS.

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Physiological effects of pyrogens Pyrogens elevate the circulating levels of inflammatory cytokines Fever Blood Coagulation Hypotension Lymphopenia Neutrophilia Elevated levels of plasma Acute phase proteins

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Low doses of pyrogens induce asymptomatic inflammatory reactions Moderate doses induce fever and changes in plasma composition

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High pyrogenic doses results in Shock characterized by cardiovascular dysfunction Vasodilation vasoconstriction endothelium dysfunction multiple organ dysfunction or failure death.

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Sources The sources of pyrogens in parenteral products include; water used at the end stages of the purification crystallization of the drug or excipients water used during processing. packaging components; chemicals, raw materials or equipment used in the preparation of the product. The presence of endotoxins on devices may be attributed to water in the manufacturing process.

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washing of components such as filter media (filters) Washing/rinsing of tubing or other plastic devices prior to their sterilization. The drug is biologically produced, incomplete removal of the microorganisms during purification.

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Measurement of pyrogens Condition: Pyrogens are generally assessed using rabbits which are stored in carefully controlled conditions and whose temperature is monitored before the administration of the test product. British Pharmacopoeia (2002) Test initially based on three rabbits; the number is progressively increased if the product fails at any one of four stages .

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Sample of the product Injected into the marginal ear vein at a dose no greater than 10 ml/kg. Monitored for the 3-hour period immediately after injection ( 30-minute intervals ) - maximum rise in temperature will be detected in this 3-hour period immediately after injection

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limitations of the rabbit pyrogen test Repeated use of animals leads to endotoxin tolerance low reactivity to the endotoxin produced by certain species, e.g. Legionella . variability in control results when identical standardized endotoxin preparations are used. Seasonal variation Inter-laboratory factors Rabbit species variations

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certain drugs may, themselves, elicit a rise in temperature on administration (diclofenac) Ex. Radiopharmaceuticals , cancer chemotherapeutic agents , hypnotics and narcotics, vitamins, steroids and some antibiotics Pyrogens may be hidden by the pharmacological activity of the product’s components ( sensitive to detect endotoxin )

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Measurement of bacterial endotoxins Advantage: Considerably more sensitive ( 1000-fold) than the pyrogen test Legionella endotoxin is easily detected by the LAL test Used for radiopharmaceuticals Limitation: LAL test only detects endotoxins of Gram-negative bacteria and not all pyrogens LAL test reagent comes from the American horseshoe crab Limulus polyphemus LAL - Limulus amoebocyte lysate Limulus polyphemus

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blood of freshly captured horseshoe crabs LAL test reagent Amoebocytes are concentrated washed and lysed with endotoxin -free water remaining cellular fragments and its activity optimized (metallic cations , pH adjustment and Additives) freeze-dried The samples of products are incubated with Limulus amoebocyte lysate at 37°C . If endotoxins are present a solid gel forms , indicating the presence of endotoxins .

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The British Pharmacopoeia (2002) describes six separate methodologies for the test for endotoxin. Gel-clot limit test Gel clot semi quantitative Turbidimetric kinetic method Chromogenic kinetic method Chromogenic end-point method Turbidimetric end-point method Coloured products cannot be test ed by turbidimetric and chromogenic methods , as precipitate formation may be mistaken for a positive response .

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The gel-clot method is most commonly used. The test is conducted by adding the LAL reagent to an equal volume of test solution , agitating and storing at 37°C for 1 hour when the end-point is determined by inversion of the tubes . If a solid clot remains intact the product is considered to contain endotoxins. Chromogenic method s utilize colorimetry but do not depend on the clottable protein . A synthetic substrate is used that contains an amino acid sequence similar to clottable protein.

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The activated proclotting enzyme cleaves a p -nitroanilide chromophore from the synthetic substrate and the colour produced is proportional to the amount of endotoxin.

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The turbidimetric LAL method: increase in endotoxin concent ration will cause a proportional increase in turbidity caused by the precipitation of the clottable protein (coagulogen). The optical density is read spectrophotometrically either at a fixed time or constantly for kinetic assays as turbidity develops.

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Chromogenic kinetic method Depend on the relationship between the logarithm of the response and the logarithm of the endotoxin concentration. Chromogenic end-point method The end-point methods relate endotoxin levels to the quantity of chromophore released .

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Chemical inhibitors ( divalent cations necessary for the reaction) inappropriate pH changes Physical inhibition Adsorption of endotox in or be caused by viscosity of the product Even the type of glassware may affect the test (Siliconized glassware or plastic can inhibit gel-clot formation ) Depyrogenated glassware must be used throughout Interfere with the interaction between LAL and endotoxin

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The presence of pyrogen in pharmaceutical product causes: Febrile reactions ( fever, chills, pain in back and legs) Pyrogen are rarely fatal.

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Glass Metal Plastic 1.Equipment Volatile Non Volatile 2.Solvent 3. Solute The sources of pyrogens

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Elimination of pyrogen Glass Metal Plastic 1.Equipment Heating to 250 o C for 45 min Heating to 650 o C for 1 min Heating with dilute. Acid, dil. Alkali or mild oxidizing agent Wash with detergent

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Volatile Non Volatile 2.Solvent Distillation Adsorption 3. Solute Recrystalization

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Pyrogens and endotoxins are difficult to remove from products once present Easier to keep components relatively endotoxin-free rather than to remove it from the final product

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Pyrogen-free water can be produced using an ultrafiltration membrane Hollow fibre polysulphone membranes can be sanitized with sodium hydroxide , which efficiently destroys pyrogens ( nominal molecular weight limit of 5000 Da should efficiently remove endotoxins) many endotoxin-producing microorganisms multiply in ambient temperature Water for Injection systems, especially reverse osmosis (RO) syste ms.

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Circulating hot water at temperatures above 75°C provides an environment that is not conducive to microbial growth and thus the formation of endotoxin. Circulating water at approximately 60°C causes some concern as some Gram-negative organisms, e.g. Legionella pneumophilia , will survive and grow at 57°C.

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Endotoxins in parenteral pharmaceuticals The limits for endotoxin are based on the dose of the product. Put simply, the endotoxin limit, EL, which represents the maximum amount of endotoxin that is allowed in a specific dose , is inversely related to the dose of the drug. (Endotoxin Limit) EL = K M

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K is the threshold human pyrogenic dose of endotoxin per kg body weight M is the maximum human dose of the product in kg body weight that would be administered in a single 1-hour period. The endotoxin limit is the level at which a product is decleared pyrogenic or non-pyrogenic

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Gel-clot reagent sensitivities are generally in the range 0.015 –0.5 EU/ml United States Pharmacopoeia ( endotoxin limits) 0.5EU/ml for Dextrose Infusion 5EU/mg promethazine in Promethazine Injection USP 10EU/mg of mitomycin in Mitomycin for Injection USP 24 EU/mg warfarin sodium in Warfarin Sodium for Injection British Pharmacopoeia ( endotoxin limits) 0.25 IU/ml in Glucose Intravenous Infusion insulin should contain 10 IU/mg of endotoxin CSF is reduced to 0.2 EU/kg ( Cerebrospinal fluid ) because the intrathecal route is the most toxic route for endotoxins.

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