For 40 years, researchers have relied on swordtail blood to detect endotoxins in pharmaceuticals. Now synthetic alternatives and updated regulations may put an end to this practice.
Each year in May, when the tide coincides with a full moon, swordtails (or horseshoe crabs) gather on the beaches of the mid-Atlantic coast to lay millions of eggs. At the same time, migratory coastal
birds flying north make a stop to feast on these eggs.
This seemingly precisely timed series of events has been going on for over ten thousand years, but in the last few decades it has been interrupted by the pharmaceutical industry needing swordtail blood.
Industry uses this blood to test the safety of drugs before distributing them to humans. Pharmaceutical companies collect more than one million swordtails each year for this purpose.
If the swordtails were to suddenly disappear, the availability of medicines would be jeopardized. "From a drug supply chain perspective, it's pretty crazy that we put ourselves in such a risky situation," says Tim Chernak, a chemist at the University of Michigan. "There could be a pandemic and we wouldn't be able to deploy vaccination if this animal just ceased to exist."
To protect this fragile ecosystem and manage risks in the drug supply chain, scientists over the past four decades have created synthetic alternatives to horseshoe crab blood for drug safety testing. Adoption of these methods has been slow, but environmental researchers are hopeful that recent changes in testing regulations will make the industry less dependent on these ancient creatures.
Scientific basis for endotoxin testing
Drug manufacturers must test all injectable drugs and medical devices, including vaccines, cell and gene therapies, and dialysis products, for endotoxins.
Endotoxin is a naturally occurring component of the cell wall of Gram-negative bacteria that provides the bacteria with structural and defense functions.
When pharmaceutical companies use these bacteria to produce recombinant protein and nucleic acid drugs, the bacteria can release endotoxins during their growth and death. The main endotoxin of concern in the pharmaceutical industry is lipopolysaccharides (LPS), and most endotoxin tests are designed to detect LPS.
"Endotoxin, once in our bloodstream, triggers many immune responses," says Jack Ling Din, a molecular immunologist and professor emeritus at the National University of Singapore. Endotoxins can cause inflammation, septic shock and, if left unchecked, death.
Photo: the-scientist.com
In 1956, medical researchers Frederick Bang and Jack Levine discovered that the blood of the swordtail coagulates upon contact with endotoxins. This clotting mechanism protects it by trapping invading microbes and their toxins, preventing them from spreading through the horseshoe crab's circulatory system.
Two decades after this discovery, the Food and Drug Administration (FDA) approved Limulus amebocyte lysate (LAL), an aqueous extract of swordtail blood containing clotting proteins, for endotoxin testing. This replaced the use of rabbits, which had been used for endotoxin testing since the 1940s.
The LAL test works as follows: if a drug is contaminated with endotoxin, it activates the Limulus enzyme clotting factor C, which then sets off a chain of reactions leading to the formation of an insoluble gel. This is the basis of the LAL gel test, but the other two LAL methods are based on either color change or fluorescence.
Toward synthetic alternatives for endotoxin testing
The development of synthetic alternatives for endotoxin testing began in the 1980s when embryos in a national in vitro fertilization (IVF) program in Singapore began to die prematurely. The suspected culprit was endotoxin.
Bow Ho, a microbiologist at the National University of Singapore, Ding's husband and co-author of many projects, used LAL tests to confirm whether embryos were contaminated with bacteria. But because of limited research funding, the cost of the LAL kit was financially prohibitive.
They considered using horseshoe crabs native to Singapore, but that too was problematic. "We couldn't get enough blood without wiping out this species," says Dean. Instead, she and Ho decided to develop a synthetic alternative to horseshoe crab blood. "We decided we had to clone the factor C gene."
Photo: the-scientist.com
She first cloned factor C in 1995, and in 2001 developed a fluorescent test based on recombinant factor C (rFC) alone to detect endotoxins. In her test, when endotoxin from Gram-negative bacteria activated rFC, it hydrolyzed the fluorogenic substrate.
The scientists found that rFC produced lower background fluorescence and was more sensitive than the commercial LAL test that existed at the time. Lonza licensed and commercialized the test in 2003.
It wasn't until 2018 that the FDA approved the first drug that utilized rFC to test for endotoxins - Emgalit (galcanezumab) by Eli Lilly and Company.
"There has been a long debate about the data and its comparability to LAL," says Jay Bolden, senior director of analytical services and quality assurance at Eli Lilly. "The fact that this is a biotechnology product was overlooked. It's exactly the same protein that's in horseshoe crab."
Eli Lilly began using rFC in 2013, when the company was collecting a lot of data on the synthetic alternative: how well it performed, how comparable the results were to LAL and whether it met the international standards set by the International Council for Harmonization of Technical Requirements for Medicinal Products for Medical Use.
Bolden, who led these studies, said the data "looked really good." In another analysis, Bolden compiled more than 1,000 data comparing LAL to rFC from the literature and found that rFC was again comparable to LAL. Other studies have also shown that endotoxin testing with rFC is equivalent to LAL.
"It's on par with LAL," Dean says. "There is no reason to produce a LAL that has some variation in sensitivity in detection."
Using purified recombinant proteins can also be more standardized compared to blood, whose composition can vary depending on the horseshoe crab's health, living conditions and age. "This type of recombinant reagent is a great example of something that can reduce the burden on many systems," Chernak says.
Photo: the-scientist.com
Since then, many other pharmaceutical companies have produced their own synthetic alternatives for endotoxin testing based on rFC or the entire swordtail blood coagulation cascade system, called recombinant cascade reagent. Despite these efforts, however, drug manufacturers remain hesitant to abandon horseshoe crab blood.
Revised recommendations after decades
Of the world's 50 largest pharmaceutical companies, only 10 to 15 have begun using synthetic alternatives to test for endotoxins, according to Elizabeth Bennett, communications director for Revive and Restore, a conservation organization that uses biotechnology to help endangered species survive.
"In general, the vast majority of pharmaceutical companies are still using LAL," Bennett said. One barrier to adoption she mentioned is regulatory guidance.
The United States Pharmacopeia (USP), an independent, non-profit organization that publishes quality guidelines for the medical industry, establishes guidelines for endotoxin testing for the United States. In the past, USP has included guidelines for the LAL test, but not for synthetic alternatives. Despite their independence from the FDA, the standards set by USP are often used by regulatory agencies.
"I view it as a prescription book," Chernak said. He mentioned that the "prescriptions" in the USP are what the FDA expects to see in a drug application. For pharmaceutical companies, using these prescribed prescriptions ensures a seamless filing.
"The fate of a product worth billions of dollars is at stake here. A pharmaceutical company cannot afford to make a mistake when submitting an application to the FDA," Chernak says. "The absence of a USP chapter on synthetic alternatives has slowed the transition considerably."
Bennett saw this firsthand when she asked pharmaceutical companies why they hadn't switched to the new method. The answer she received was always the same: "Regulatory regulations won't let me do it," she heard each time.
Work to update USP recommendations for endotoxin testing began in 2019, but these early attempts failed for a variety of reasons - internal politics, LAL suppliers' interest in continuing to manufacture LALs, and lingering doubts about alternatives.
It wasn't until May 2025 that USP added a new chapter on synthetic alternatives to LAL. Bolden, who served on USP's Microbiology Expert Committee, helped develop this new chapter starting in 2023.
USP chapters are vetted at the subcommittee level - such as the endotoxin subcommittee - by the full expert committee, as well as stakeholders including consultants, pharmaceutical company representatives, FDA and USP representatives before they are approved.
While the USP guidelines do not require that the LAL test be replaced by the rFC, this is a significant step forward in recognizing the rFC as an equivalent test.
Celebrating sustainable development
"Revive and Restore recently launched a sustainability assessment system for endotoxin testing that tracks pharmaceutical companies' adoption of sustainable alternatives to LALs.
These scorecards are what Bennett called "the first public indicator that measures the pharmaceutical industry's progress in sustainable endotoxin testing. It's an accountability tool. We like to say it's the gaming version of environmental stewardship that we'd like to see in the industry."
"Revive and Restore has worked with various pharmaceutical companies to develop scorecards that measure public acceptance of LAL substitution, reduction in LAL use, and whether pharmaceutical companies have adopted synthetic alternatives for new or legacy products."
"We need to determine what is really right for the pharmaceutical industry beyond the ethical and environmental standpoints," says Bennett. She believes that the best outcomes for these companies are likely to come from improving their supply chains and optimizing processes and business operations.
Photo: the-scientist.com
"rFC has benefited us in terms of quality. It has secured our supply chain. It's obviously better from a biodiversity and ethical perspective, and for us, frankly, it's been cost-effective," says Bennett. "We really wanted this to be an opportunity for pharmaceutical companies to be recognized for what they do, because how often are big pharma companies celebrated in popular culture? Not often."
"This is the oldest creature on our planet. It has survived five mass extinctions. It's been on the planet for 450 million years," Chernak said. "We have no reason to continue to prey on this animal."