A new era in pyrogen testing

Abstract

Pyrogens are substances (usually of biological origin) that cause fever after injection. The best-studied pyrogen is lipopolysaccharide (LPS, also known as endotoxin), found in the membrane of Gram-negative bacteria. During Gram-negative sepsis, endotoxin stimulates host macrophages to release inflammatory cytokines and excessive inflammation causes multiple organ failure and death. Endotoxins are thus ubiquitous pathogenic molecules that are a bane to the pharmaceutical industry and medical community. Limulus amoebocyte lysate (LAL) has been widely used for ∼25 years for the detection of endotoxin in quality control of injectable drugs and medical devices. However, variations in sensitivity and specificity of LAL to endotoxin, and the limited supply of limulus (horseshoe crabs) has called for an alternative pyrogen test. Recombinant Factor C (rFC), the endotoxin-inducible coagulation enzyme in LAL, forms the basis of a novel micro-enzymatic assay for high-throughput screens of endotoxin and opens a new era in endotoxin testing. Endotoxin activates the rFC zymogen, which catalytically hydrolyses synthetic substrates to form measurable products, thus quantifying the endotoxin.