During the early days of the pharmaceutical industry it was noticed that some solutions when injected into the bloodstream induced fevers. Investigations found that almost all of these fevers were associated with a group of contaminants termed pyrogens (‘heat or fever generating’). These were classified as either exogenous or endogenous pyrogens.
Exogenous pyrogens are fever causing materials found in the environment, of these – endotoxins are the most researched and are lipospolysacchrides (LPS), found in the outer membrane of the cell wall of Gram negative microorganisms, they are heat stable and can cause severe patient reactions when present in parenterals or medical devices.
An “endotoxin” is a toxin that is a structural molecule of the bacteria that is recognized by the immune system.
Endotoxin is heat resistant and stable in most environmental conditions. For example, it can only be destroyed at high temperatures (185°C for 3 hours or 250°C for 30 min.); by aggressive chemicals such as NaOH or H2O2; or removed by expensive chromatographic processes. Consequently, it can be a major problem source in contamination control.
Endotoxin toxicity is not dependent upon a living cell, heat sterilization or other chemical/physical processes are ineffective control measures as killing the cells actually releases ‘free’ endotoxin from the cell wall.
Endotoxin control is a critical quality requirement in the manufacture of parenterals, biologicals, medical devices, and in the operation of dialysis centers, blood donor centers, and in other areas specifically regulated for endotoxin content and the prevention of endotoxin contamination.
As all mammals can be affected by endotoxins (although sensitivity levels vary) one of the first tests used to determine if endotoxins were present, was the Rabbit Pyrogen Test (RPT). A rabbit was inoculated with the test substance and then monitored it to see if a fever was induced. This test however does not give a quantitative result, is time consuming and is not suitable for products that may in themselves adversely effect the animal.
The most commonly used approach now is a Limulus Amoebocyte Lysate or LAL test. The LAL test focuses in particular on 2-keto-3-deoxyoctonoic acid – and it is this which is used as an indicator in the majority of endotoxin assays.
LAL is a reagent derived from the blood cells of the horseshoe crab, unlike a mammal the crab does not have a developed immune system, however the LAL component in its blood will bind to and inactivate endotoxins in the crab the resulting clot also forms a protective barrier against bacterial infection.
At its simplest the LAL test consists of adding LAL reagent to the sample in a test tube, incubating at 37°C for 1 hour. The tube is then gently inverted – if a gel or clot has formed then a positive result is recorded.
Although a positive LAL test indicates the presence of pyrogens a negative result does not necessarily mean the product is pyrogen free, as non- endotoxin pyrogens may be present but not picked up by the LAL test. But this test does enable products that are unsuitable for the rabbit test to be screened.
Endotoxin results are expressed as Endotoxin Unit (EU) per gram or mg or International Unit (IU) per gram or mg; 1 EU = 1 IU.