Every year in May when high tide coincides with the full moon, horseshoe crabs convene on the beaches of the mid-Atlantic coast to lay millions of eggs. At the same time, migratory shorebirds on their journey north make a pit stop to feed on these eggs. This seemingly precisely timed series of events has been happening for over ten thousand years, but for the last few decades, it has been interrupted by the pharmaceutical industry seeking horseshoe crab blood. The industry uses this blood to test the safety of the drug supply before they can distribute the therapies to people. Drug companies harvest more than one million horseshoe crabs each year for this purpose.
If these crabs suddenly disappeared, medicine availability would be in trouble. “It’s kind of crazy from a drug supply chain perspective that we’ve put ourselves in this really risky situation,” said Tim Cernak, a chemist at the University of Michigan. “We could have a pandemic, and we would be unable to deploy a vaccine if that animal just stopped existing.”
Tim Cernak works at the intersection of chemistry and conservation.
Adam Kanzer
To protect this fragile ecosystem and to manage risk in the drug supply chain, scientists have created synthetic alternatives to horseshoe crab blood to test drug safety over the last four decades. Adoption of these methods has been slow, yet scientists and conservationists are hopeful that recent changes to testing guidelines will make the industry less reliant on these ancient creatures.
The Science Behind Endotoxin Testing
Drug manufacturers need to test every injectable drug or medical device—including vaccines, cell and gene therapies, and dialysis products—for endotoxins. Endotoxins are a natural component of the cell wall of Gram-negative bacteria that provide the bacteria with a structural and protective function. When drug companies use these bacteria to produce recombinant protein and nucleic acid drugs, the bacteria can release endotoxins as a normal part of their growth and death. The main endotoxin of concern for the drug industry are lipopolysaccharides (LPS), and most endotoxin testing is designed to detect LPS.
“Endotoxin, when it enters into our bloodstream, will switch on a lot of immune responses,” said Jeak Ling Ding, a molecular innate immunologist and emeritus professor at the National University of Singapore. Endotoxins can trigger inflammation, septic shock, and if not controlled, death.
In 1956, medical researchers Frederick Bang and Jack Levin found that horseshoe crab blood would clot if it came into contact with endotoxins. This clotting mechanism protects the crab by trapping invading microbes and their toxins, preventing their spread through the crab’s circulatory system.1 Two decades after this discovery, the Food and Drug Administration (FDA) approved limulus amebocyte lysate (LAL)—the aqueous extract of blood from horseshoe crabs containing clotting proteins—for endotoxin testing. This replaced the use of rabbits, which had been used for endotoxin testing since the 1940s.2
The LAL test works like this: If a drug is contaminated with endotoxin, it will activate the enzyme limulus clotting Factor C, which then triggers a cascade of reactions that result in an insoluble gel. This is the basis of the gel clot LAL test, but two other LAL methods rely on either a color change or a fluorescence readout.
Toward Synthetic Alternatives for Endotoxin Testing
The development of synthetic alternatives for endotoxin testing began in the 1980s when embryos in Singapore’s national in vitro fertilization (IVF) program began dying prematurely. The suspected culprit was endotoxin. Bow Ho, a microbiologist at the National University of Singapore and Ding’s husband and co-investigator on many projects, was using LAL tests to confirm whether the embryos were infected with bacteria. But with limited research funding, the cost of the LAL kit was not financially sustainable. They considered turning to horseshoe crabs found in Singapore, but this too would be problematic. “There was no way we could get enough blood and not kill the species," Ding said. Instead, she and Ho aimed to pioneer a synthetic alternative to horseshoe crab blood. “We decided that we should clone the gene for Factor C.”
Jeak Ling Ding spearheaded the development of an alternative assay to LAL in the 1990s.
National University of Singapore
She first cloned Factor C in 1995 and later developed a fluorescence-based assay in 2001 that relied on just recombinant Factor C (rFC) to detect endotoxins.3,4 In her test, when endotoxin from Gram-negative bacteria activated rFC, it hydrolyzed a fluorogenic substrate. They found that rFC produced a lower background fluorescence and was more sensitive than the commercial LAL test at the time. Lonza Inc. licensed and commercialized the test in 2003. It wasn’t until 2018 that the first drug that used rFC for endotoxin testing—Eli Lilly and Company’s Emgality™ (galcanezumab)—was approved by the FDA.
“There was a long debate around the data and the comparability of the data [to LAL],” said Jay Bolden, senior director of the Analytical Services and Quality Control Organization at Eli Lilly. “What got lost in that is that it's the product of biotechnology. It's the exact same protein that's in the horseshoe crab.”
Eli Lilly got its start with rFC in 2013 when the company started generating a lot of data on the synthetic alternative: how well it worked, how comparable the readout was to LAL, and whether it would meet international standards set by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Bolden, who spearheaded these studies at Lilly, said that the data “looked really good.”5 In another analysis, Bolden compiled more than 1,000 data points comparing LAL to rFC from the literature and found that rFC was again comparable to LAL.6 Other studies also showed that endotoxin testing with rFC was equivalent to LAL.2,7
“It stands shoulder to shoulder with LAL,” said Ding. “There is no reason anymore to produce LAL which has some variations of sensitivity in detection.”
Using purified recombinant proteins also has the potential to be more standardized as compared to blood, the composition of which can change due to the crab’s health, living environment, and age. “This kind of recombinant reagent is a perfect example of something that could relieve stress in a lot of different systems,” said Cernak.
Since then, many other pharmaceutical companies have been producing their own synthetic alternatives for endotoxin testing based on rFC or the entire horseshoe crab coagulation cascade, called the recombinant cascade reagent. Yet despite these efforts, drug manufacturers have remained hesitant to turn away from horseshoe crab blood.
Revised Recommendations Decades Later
Of the top 50 pharmaceutical companies in the world, only 10 to 15 of them have started using synthetic alternatives for endotoxin testing, said Elizabeth Bennett, the director of communications at Revive & Restore, a conservation organization that applies biotechnology towards the survival of endangered species. “By and large, a vast majority of pharmaceutical companies are still using LAL,” Bennett said. One barrier to adoption she cited is the regulatory guidelines.
Elizabeth Bennett led the Sustainability Scorecard project at Revive & Restore.
Revive & Restore
The US Pharmacopeia (USP), an independent nonprofit organization that publishes quality guidelines for the medical industry, sets the endotoxin testing guidelines for the United States. In the past, USP included guidelines for the LAL test, but not synthetic alternatives. While independent from the FDA, standards set by the USP are often used by regulatory agencies.
“I view it like a recipe book,” said Cernak. He mentioned that the “recipes” in the USP are what the FDA expects to see in a drug submission. For drug companies, using these prescribed recipes ensures a smooth submission. “What’s at risk here is the fate of a multi-billion-dollar product. A pharmaceutical company can’t afford to mess up their submission to the FDA,” said Cernak. “The absence of a USP chapter [on synthetic alternatives] created a lot of slowing down of the transition.” Bennett saw this firsthand when she asked pharma companies why they haven’t made the switch. The response she received was always, “the regulatory guidelines did not say I could,” she said.
Efforts to update the USP guidelines on endotoxin testing began in 2019, but these early attempts never panned out for various reasons—internal politics, a vested interest in continued LAL production by LAL vendors, and lingering doubts on the alternatives. It wasn’t until May 2025 that the USP added a new chapter on synthetic alternatives to LAL. Bolden, who was part of the USP Microbiology Expert Committee, helped draft this new chapter beginning in 2023. USP chapters undergo review by subcommittee level—for example, the endotoxin subcommittee—the full expert committee, and stakeholders include consultants, pharmaceutical representatives, the FDA, and USP representatives before they are approved.
While USP guidelines don’t call for a replacement of the LAL test with rFC, it is a significant step forward in recognizing rFC as an equivalent test. “We’re just so excited that finally the USP…has accepted recombinant Factor C as a compendial,” said Ding.
Celebrating Sustainability
Revive & Restore recently launched the Sustainability Scorecards for Endotoxin Testing, which track the adoption of sustainable alternatives to LAL by pharmaceutical companies. These scorecards are what Bennett called the “first public metric that measures the progress of the pharmaceutical industry” in sustainable endotoxin testing. She added, “It’s an accountability tool. We like to say [it’s] a gamified version of the environmental stewardship that we would like to see in the industry.”
Revive & Restore collaborated with the Horseshoe Crab Recovery Coalition and various pharmaceutical companies to develop the scorecards, which measure public acknowledgement of replacing LAL, reducing LAL use, and whether pharmaceutical companies have adopted synthetic alternatives for new or legacy products.
“We need to define what the right thing actually is for pharma, beyond an ethical standpoint, beyond an ecological standpoint,” said Bennett. She thinks that the best outcomes for these companies are likely to involve improving their supply chains and streamlining their processes and business operations.
Over the last decade, Jay Bolden and his team at Eli Lilly have generated over one thousand data points comparing LAL to rFC.
Eli Lilly and Company/Todd Rosenburg
This is exactly what Bolden said about Eli Lilly, which sits at the top of the leaderboard. “[rFC has] benefited us from a quality perspective. It’s secured our supply chain. It’s better obviously just from a biodiversity and ethical perspective, and then for us, frankly, it’s been cost-effective,” he said.
Bennett added, “We really wanted it to be an opportunity for those pharma companies to get some recognition for what they’re doing, because how often is big pharma celebrated in popular culture? Not much.”
Other companies in the industry that top the scoreboard are GSK, Amgen, and Sanofi, with many other companies following suit in using synthetic alternatives for endotoxin testing.
“This is the most ancient creature on our planet. It survived five mass extinctions. It’s been on the planet for 450 million years,” said Cernak. “There’s no reason for us to continue to harvest this animal.”
- Muta T, et al. Horseshoe crab coagulation Factor B. A unique serine protease zymogen activated by cleavage of an Ile-Ile bond. J Biol Chem. 1993;268(28):21384-21388.
- Maloney T, et al. Saving the horseshoe crab: A synthetic alternative to horseshoe crab blood for endotoxin detection. PLoS Biol. 2018;16(10):e2006607.
- Ding JL, et al. Molecular cloning and sequence analysis of factor C cDNA from the Singapore horseshoe crab, Carcinoscorpius rotundicauda. Mol Mar Biol Biotechnol. 1995;4(1):90-103.
- Ding JL, Ho B. A new era in pyrogen testing. Trends Biotechnol. 2001;19(8):277-281.
- Bolden J, Smith K. Application of recombinant factor C reagent for the detection of bacterial endotoxins in pharmaceutical products. PDA J Pharm Sci Technol. 2017;71(5):405-412.
- Bolden J, et al. Currently available recombinant alternatives to horseshoe crab blood lysates: Are they comparable for the detection of environmental bacterial endotoxins? A review. PDA J Pharm Sci Technol. 2020;74(5):602-611.
- Kang DH, et al. A study on the application of recombinant factor C (rFC) assay using biopharmaceuticals. Microorganisms. 2024;12(3):516.
