Pyrogen, Endotoxin, Limulus Amebocyte Lysate (LAL) and Bacterial Endotoxin Testing (BET) are all used interchangeably when referring to the testing performed in the microbiology laboratory. Bacterial endotoxins are very potent inflammatory agents even after sterilization and consist of a polysaccharide (sugar) chain and a lipid moiety, known as lipid A, which is responsible for producing fever, a lowering of the blood pressure, and activation of inflammation and coagulation in patients exposed to these toxins. Firms operating in the medical device, pharmaceutical and biotech industries must control production environments and limit product exposure to sources of endotoxin from raw material receipt through the end of the manufacturing process and comply with global regulatory requirements surrounding endotoxin levels in finished product contacting either the cerebral spinal fluid or cardiovascular system. Standard practice in these industries is to demonstrate compliance through Bacterial Endotoxin (LAL) Testing as a product release test.
MCL employs 3 LAL test methodologies in our microbiology laboratory. They are the Gel-Clot, Kinetic Turbidimetric and Chromogenic LAL methods.
The Gel-Clot method is a qualitative (Pass/Fail) LAL test. It is an excellent choice for customers who submit a very limited number of samples, perform incoming quality control of raw materials or where a quantitative endotoxin result is not necessary. In the Gel-Clot method, endotoxin triggers a cascade of enzymatic reactions, which result in activated clotting enzyme. The formation of the gel clot is proportional to the concentration of the endotoxin in the sample. Although still considered the referee test for determining endotoxin concentrations, many customers choose to go with a quantitative method described below.
The kinetic turbidimetric method is a quantitative LAL test which can precisely measure endotoxin concentrations of samples. The kinetic turbidimetric method is the most sensitive of the LAL assays, having sensitivity as low as 0.001 EU/ml. The Kinetic Turbidimetric method is the general choice when considering cost, speed and sensitivity. In the kinetic assay, the proenzyme in the LAL is activated when in contact with endotoxins. The rate of activation is directly proportional to the concentration of endotoxin present in the sample. The level of activation can be precisely quantified spectrophotometrically.
The chromogenic method is another quantitative, kinetic assay for the detection of bacterial endotoxins. A sample is mixed with the LAL/Substrate reagent, placed in an incubating plate reader and monitored over time for the appearance of a yellow color. It is the chromogenic nature of this assay that makes it the most appropriate choice for testing small volume parenterals, vaccines, antibiotics and biologicals. The chromogenic method is less affected by inhibitory substances and materials that may interfere in the turbidimetric and gel-clot assays.
The founders of MCL bring years of experience performing all methods of endotoxin testing on drug substances, drug products and medical devices. Employing a proven and accredited ISO 17025:2011 quality system and lean mentality, MCL will bring the highest level of quality, attention and detail to our clients in assuring that their endotoxin testing will be successfully executed in the most cost effective and timely manner. The founders of MCL are available for consultation on test method selection, specific laboratory procedures or for challenging types of products.