Testing for Bacterial Endotoxins

Testing for endotoxins (a toxin present in a bacteria cell that is released when the cell disintegrates) in injectable biologics was revolutionized in Building 29A by Drs. Edward Seligmann and H. Donald Hochstein. Endotoxins are part of the outer membrane of the cell wall of Gram-negative bacteria and had been discovered to be the cause of fevers in some patients receiving injections.

Pyrogens are endotoxins that cause fevers. Building on previous studies by scientists at Johns Hopkins University, Drs. Seligmann and Hochstein developed a new calibrated test, Limulus amebocyte lysate (LAL), for pyrogens using horseshoe crab blood instead of rabbits’ ears. For thirty years, the standard test had been to inject rabbits’ ears and measure the reaction. This new LAL test was more sensitive, rapid, and economical. The LAL became the standard worldwide test for bacterial endotoxins.

This testing was conducted in the Pyrogens Section of the National Institutes of Health (NIH) Division of Biologics Standards (DBS) Laboratory of Control Activities, which was located in Building 29, fourth floor from 1960 to 1967, and then moved to Building 29A, first floor when the annex building opened in 1967.

The important test for pyrogens in biologics as a measure of purity and integrity of the biological product was elevated technologically in research the FDA Bureau of Biologics pursued in 1980. For products derived from bacteria, pyrogens can be found in either the cell wall of the bacteria (an “endotoxin”) or from within the cell (an “endogenous toxin”). Emergence of the so-called “Limulus amoebocyte lysate” (LAL) test changed the sensitivity of pyrogenic analysis. Traditionally, the material to be tested was injected into a rabbit to check for the presence of an endotoxin. However, pyrogens are not always pyrogenic for both humans and rabbits. In the LAS test procedure, if a pyrogen is present it will produce a temperature when the test substance is treated and then administered to the rabbit. The LAL test, with radioimmunoassay, can also be used for endogenous pyrogens.