How do endotoxin tests work?

Bacteria can be found everywhere—even sometimes in clean rooms. That’s a problem for manufacturers of drugs and medical devices that are injected or implanted. Even if manufacturers can remove the microbes themselves, molecular traces can remain. If they make their way into the body, these toxins activate a powerful immune response, causing fever and inflammation.

To make sure these microbial signatures, called pyrogens, are absent from injected drugs, manufacturers test ingredients, containers, water systems, and finished drug products for specific molecules in . The longest-used assay depends on ingredients from wild animals, but regulators are now approving the use of animal-free alternatives. Here’s how these tests work.

LAL: The legacy test

A pyrogen is any molecule that can cause a fever if injected. One subset of pyrogens are lipopolysaccharides (LPS), also called endotoxins. These glycolipids, found in the outer membrane of gram-negative bacteria, comprise lipid A, a core oligosaccharide, and a repeated outer sugar chain called the O antigen. Most gram-negative bacteria have LPS, although the sugar modifications can vary. Still, the lipids’ ubiquity makes them a good target for many immune systems—including that of the horseshoe crab Limulus polyphemus.

“In the horseshoe crab blood, the presence of endotoxin . . . triggers a chain reaction,” says Michael Reynier, a senior vice president at the diagnostics firm bioMérieux. The reaction starts with a protein called factor C, which binds to LPS. This binding changes factor C’s shape and activates its protease abilities. Now active, the enzyme cleaves itself and then other proteins, in turn activating their protease functions. By the end of this game of proteolytic telephone in the horseshoe crab, a cleaved protein forms a clot that helps trap bacteria.

The pharmaceutical industry has adopted crab blood for its own purposes. To make sure their products don’t contain endotoxins, drugmakers mostly use a reagent called limulus amoebocyte lysate (LAL), made from extracted crab blood. While labs once measured LAL activation through a clotting assay, most LAL assays today use a synthetic substrate that turns yellow if it is cleaved by the final protease in the signaling system. The color change, measured by absorbance, alerts manufacturers to the presence of LPS.

“It’s detecting contamination at the picogram–femtogram level,” says Jay Bolden, a microbiologist at Eli Lilly and Company. “It’s a very, very sensitive assay.”

The LAL assay has some problems, however. One is high batch-to-batch variability. Another is that it contains a protease that can cause false-positive results when drug products are run through cellulose filters. Finally, and most importantly to many advocates, LAL relies on blood harvested from wild animals.

Recombinant reagents

Decades ago, scientists looking for an alternative to animal testing cloned factor C, the horseshoe crab protein that senses LPS. That recombinant factor C (rFC) is now also used in pharmaceutical quality tests.

In extracted crab blood and LAL, rounds of proteolysis amplify factor C’s signal: one activated factor C molecule can activate an avalanche of dozens or hundreds of downstream molecules, helping researchers spot tiny quantities of endotoxins. To detect a signal from rFC without that amplification, scientists designed a new synthetic substrate that fluoresces when rFC cleaves it. Fluorescence signals allow researchers to identify a small signal because they contrast with background noise more than changes in visible light absorbance do.

The sensitivities of rFC and LAL reagents are equivalent, even though the readouts are different, bioMérieux’s Reynier says. “The sensitivity level of both are clearly more than enough to detect any trace of contamination.” Regulators in Europe and China have fully accepted rFC tests, and similar acceptance is under way in the US. Regulators in other countries are considering the tests.

But fluorescent assays also necessitate different equipment. Bolden says that for some labs, it’s “a big barrier . . . to have to switch from an absorbance technology to a fluorescence detection technology.”

A newer riff on the endotoxin tests is recombinant cascade reagents (RCRs), which contain more components of the proteolytic cascade found in LAL—but these proteases are manufactured rather than harvested from wild crabs. RCRs include rounds of proteases downstream of factor C but exclude the enzyme that senses β-glucans, such as those found in the cellulose in some filters. This proteolytic amplification allows scientists to monitor the signal with a color change—a selling point for some customers because the readout is the same as using LAL.

At least three RCRs are now available, from the LAL producers Charles River Laboratories and Associates of Cape Cod and from Xiamen Bioendo Technology, which also sells an LAL equivalent made from Asian horseshoe crabs. Manufacturers interested in leaving LAL behind are still figuring out whether they prefer rFC or RCR tests. According to an emailed statement from Merck & Co., a recent conference on recombinant endotoxin assays included studies that “highlighted crucial differences between the various recombinant reagents.”

Beyond endotoxins

Some experts advocate testing for more than endotoxins. Helena Champion, a pharmaceutical quality consultant, says, “A great many people are unaware” that endotoxin testing overlooks molecular signatures from bacteria that have no outer membrane and those from molds, fungi, and viruses—all of which may cause fevers.

Researchers have developed technologies that recognize a wider range of pyrogens. For example, the monocyte activation test uses immune cells from human donors’ blood. These cells express many receptors to detect pyrogenic molecules—just like the surveillance cells that respond to pyrogens in people.

Champion calls the monocyte activation test “far preferable” to endotoxin testing. But while regulators in Europe, Japan, and India recognize it, the test is not yet in the pharmacopoeia in the US.