Horseshoe Crab Blood: Synthetic Alternatives to Preserve Vital Drug Testing

Horseshoe crab blood is used to produce limulus amebocyte lysate (LAL), a substance used to test for toxic endotoxins in intravenous drugs. These toxins are ubiquitous in the environment and cannot be removed through sterilization. Injection fever, a severe reaction resulting from the detection of endotoxins by LAL, was a significant 20th-century medical safety breakthrough.

However, critics are raising questions about environmental impacts and the review and approval process for synthetic alternatives. A white paper examining social, political, and economic issues associated with using horseshoe crabs to produce LAL aims to address complex problems that cut across multiple agencies and require attention to both nature and human health. The rabbit pyrogen test, created by biochemist Florence Seibert in the 1920s, required injecting intravenous drugs into rabbits and monitoring the animals to detect contaminated drugs.

The Limulus aeruginosa (LAL) method was discovered by pathobiologist Frederik Bang and medical researcher Jack Levin in the 1950s and ’60s at the Marine Biological Laboratory in Woods Hole, Massachusetts. They discovered that horseshoe crabs’ blue blood coagulated in a curious manner, leading to the discovery of endotoxin as the coagulant. LAL was refined by academic researchers, biomedical companies, and the U.S. Food and Drug Administration, and by the 1990s, it was the FDA-approved method for testing medicines for endotoxin.

Producing LAL requires harvesting horseshoe crabs from oceans and beaches, draining up to 30% of their blood in a laboratory, and returning the live crabs to the ocean. The number of crabs killed in the process and potential harm to survivors is disputed, but there are five FDA-licensed LAL producers along the U.S. East Coast, whose production and sales value are proprietary.

The population of Limulus is neither strongly growing nor declining, but the most recent federal assessment found the population is neither strongly growing nor declining. Conservationists are concerned not only about the crabs but also about the millions of shorebirds who migrate along the Atlantic coast and stop in spring to feed on horseshoe crab eggs.

Red knots were listed as threatened under the Endangered Species Act in 2015 due to horseshoe crab fishing, which threatened this key food source. Conservation groups are calling on the Livelihood-Assisted Detection (LAL) industry to find new sources for medicines, such as penicillin, which was originally produced from molds. Synthesizing these substances in laboratories offers many benefits, including being cheaper, more efficient, and avoiding species risk.

In the 1990s, researchers at the National University of Singapore invented and patented the first synthetic, endotoxin-detecting compound using horseshoe crab DNA and recombinant DNA technology. This compound, dubbed recombinant Factor C (rFC), mimics the first step in the three-part cascade reaction when LAL is exposed to endotoxin. Later, several biomedical firms produced their own versions of rFC and compounds called recombinant cascade reagents (rCRs), which reproduce the entire LAL reaction without using horseshoe crab blood.

LAL remains the dominant technology for detecting endotoxins in medicine, as the U.S. Pharmacopeia considers rFC and rCR as “alternative” methods for detecting endotoxins, requiring case-by-case validation for use. While some large pharmaceutical companies have committed to switching from LAL to rFC, most drug producers remain with the tried-and-true method. Conservation groups want the U.S. Pharmacopeia to fully certify rFC for use in industry with no extra testing or validation, while LAL producers counter that they are doing due diligence to protect public health.

Major LAL producers are now offering recombinant products, indicating a shift towards Limulus-free testing for endotoxins. Atlantic fisheries regulators are considering new harvest limits for horseshoe crabs, while the U.S. Pharmacopeia is evaluating recombinant alternatives. Public comments will be solicited in winter 2024, followed by FDA review.

Progress could be made by collecting more data on horseshoe crab populations, requiring more transparency from the LAL industry, and directing medical companies to use recombinant products for testing during the manufacturing process. Incremental actions that protect human health and the environment could be crucial steps forward.

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