Endotoxin Detection Methods — Where are we now? Tuesday, August 25, Kalavati Suvarna, Ph. Abstract Endotoxins are considered as the major contributors to the pyrogenic response observed with contaminated pharmaceutical products. Products manufactured by bioprocessing are microbiologically controlled to ensure that the end product meets the requirements for release.
Background[ edit ] Fred Bang reported in that gram-negative bacteria, even if killed, will cause the blood of the horseshoe crab to turn into a semi-solid mass. It was later recognized that the animal's blood cells, mobile cells called amoebocytescontain granules with a clotting factor known as coagulogen; this is released outside the cell when bacterial endotoxin is encountered.
The resulting coagulation is thought to contain bacterial infections in the animal's semi-closed circulatory system. In the U. Prior to that date, a much slower and more expensive test on rabbits had been used for this purpose.
This releases the chemicals from the inside of the cell the "lysate"which is then purified and freeze-dried. To test a sample for endotoxins, it is mixed with lysate and water; endotoxins are present if coagulation occurs. The primary application for LAL is the testing of parenteral pharmaceuticals and medical devices that contact blood or cerebrospinal fluid.
All assays, independent of methodology are standardized using endotoxin in water. Therefore, unless the sample is water, some components of the solution may interfere with the LAL test such that the recovery of endotoxin is affected. If the product being tested causes the endotoxin recovery to be less than expected, the product is inhibitory to the LAL test.
Products which cause higher than expected values are enhancing. Overcoming the inhibition and enhancement properties of a product is required by the FDA as part of the validation of the LAL test for use in the final release testing of injectables and medical devices.
Proper endotoxin recovery must be proven before LAL can be used to release product. It is based on the Limulus clotting factor C protein, produced by genetically modified insect gut cells.
The adoption of this test was slow, which began to change in when the European Pharmacopeia listed this test as an accepted bacterial-toxin test.interferences.
Therefore, the LAL assay requires a well-controlled and validated methodology for its correct implementation. The endotoxin In the chromogenic LAL test, the presence of endotoxin catalyzes.
the conversion of a pro-enzyme to enzyme (active form) which leads. Test 1 can be done once and need not be repeated until bacterial endotoxin standard and lysate lots have changed.
Test 2 is conducted to identify any potential interference of the test. USP’s Microbiology Expert Committee is responsible for general chapter Bacterial Endotoxin Test.
The Committee has engaged in frequent discussions regarding the assay interference issue, commonly known as Low Endotoxin Recovery (LER), since it was first reported in (1).
Interference due to the presence of organic substances with chelating or anticoagulant properties, which prevents gel formation indicative of the amount of endotoxins. An example of . The blood endotoxin level and endotoxin localization in the rat were determined before and at 6, 12 and 24 h after intraperitoneal injection of g/kg of fresh rat feces.
of endotoxin in the sample. Components of the test sample could cause interference with any of the steps in the cascade, therefore affecting the final result. Although there are some products that will cause LAL assay enhancement, the majority of LAL interference is due to .