Endotoxin

Endotoxin is lipopolysaccharide (LPS), a molecule from the outer membrane of gram-negative bacteria such as E. coli. When the bacteria die, the LPS stays behind. In the bloodstream it triggers an immune response, and enough of it causes fever, chills and, at high doses, a drop in blood pressure. Substances that cause fever are called pyrogens, and endotoxin is the most important one in drug manufacturing.

Sterile and endotoxin-free are different claims

Sterility means no living organisms. Endotoxin is not alive. The FDA's inspection guide states that endotoxins "are not removed by sterilizing or microbiological filters". A product can be filtered, pass a sterility test and still carry endotoxin from bacteria that grew in it earlier. Endotoxin is also heat-stable: the same guide notes that depyrogenating glassware has been done by heating to 250 C for 45 minutes, far beyond normal sterilisation.

So an injectable needs two separate results: a sterility test and an endotoxin test. One does not stand in for the other.

How it is measured

The standard method is the bacterial endotoxins test using limulus amebocyte lysate (LAL), a reagent made from horseshoe crab blood cells that clots in the presence of endotoxin. Recombinant versions of the reagent now exist. Results are reported in endotoxin units (EU).

The limit for a product is set by a formula, K/M:

Before the LAL test, pyrogens were detected by injecting rabbits and watching for fever. The FDA guide describes K as "the approximate threshold pyrogen dose for humans and rabbits", which is where the number comes from.

For a 70 kg adult, K works out to 350 EU in an hour. The limit per milligram of product is that budget divided by the dose, so a product given in larger amounts needs a stricter per-milligram limit. A certificate of analysis that reports endotoxin should give a number and a unit, such as EU/mg, and the limit it was tested against. A line that reads "pass" with no number, no method and no limit tells you much less.

Why this matters for peptides

Approved injectables are made under manufacturing rules that require endotoxin testing on every batch. Peptides sold outside that system may or may not have been tested, and a purity figure does not answer the question. A 99 percent pure peptide by HPLC can still carry endotoxin, because HPLC measures related peptide impurities, not bacterial fragments. The purity testing page lists which tests answer which questions.

Signs of a pyrogenic reaction after an injection are fever, chills, aches and feeling unwell within hours. These are worth reporting to a clinician, along with the batch.

The vial tracker in the app keeps the batch and source with each vial, and the dose log records any reaction against the dose and vial it followed.

Related terms: purity testing, certificate of analysis, HPLC, lyophilised. Back to the glossary.