Horseshoe Crab Blood Has Long Helped Us Make Safe Medicines. Now, Alternatives That Spare the Ancient Creatures Might Be Breaking Through

Each May, hundreds of thousands of horseshoe crabs climb onto beaches along the eastern coast of the United States. An invertebrate’s “Love Island,” males compete to procreate with the largest females. The hum of scampering claws and tapping shells fills miles of beach.

The American horseshoe crab is not the most glamorous or endearing animal. With its long, spiky tail, helmet-shaped shell and ten eyes, the 445-million-year-old species did not get this far relying on its good looks. But in this annual event, called a mass spawning, the crabs leave behind tens of thousands of eggs, which contribute to a wider ecosystem along the Eastern Seaboard and allow the ancient species to continue thriving.

It’s “the most magical natural phenomenon,” says Will Harlan, who acts as the Southeast director at the Center for Biological Diversity. He took his kids to witness a horseshoe crab mass spawning in Delaware last spring. Usually, his children are glued to their screens, says Harlan, but that night, they couldn’t look away from the sea of crabs scuttling ashore under a full moon.

Since the 1980s, humans have prized horseshoe crabs for another reason: The invertebrates’ light blue blood has likely benefitted anyone who has ever received a vaccine or flu shot. Equipped with a system that detects toxins as soon as they enter the crabs’ bloodstream, the blood is a key ingredient in a product that can detect contaminants in medicines. Pharmaceutical companies use the mechanism to ensure their drugs are safe. But amid concerns for the horseshoe crab population, conservationists, biotechnology experts and drug manufacturers alike have spent decades pushing for the adoption of synthetic alternatives that don’t harm the animal.

This spring, that effort reached a new milestone. For the first time, the industry’s primary regulatory body, the U.S. Pharmacopeia, officially recognized the alternatives as an option for companies to test vaccines. Now, 11 major pharmaceutical companies have reported an initial shift or intent to shift to these alternatives in a survey conducted by conservation groups. And the biggest producer of the blood-derived product publicly backed the crabless alternatives this summer.

Harlan says his organization, which worked on the survey of the pharmaceutical companies, hopes to see all 50 of the largest companies by profit shift to these alternatives. But it might not be a rapid transition, as making that change involves navigating an intricate network of corporations and regulatory bodies.

Around the time Harlan took his kids to Delaware Bay, Allen Burgenson took the day off work to visit another beach and flip horseshoe crabs that had gotten stuck on their backs during the spawning—a tradition he’s had since he was 3 years old. This small act can save the invertebrates from an immobile death sentence on the shores of the bay, he says. Burgenson has spent his career in biotechnology researching, advising and working on the horseshoe crab blood tests and alternatives.

Jay Bolden, too, traveled to Delaware Bay last spring as part of a meeting of leaders in the pharmaceutical industry. Bolden spearheaded the first widespread shift to the crabless alternatives in the U.S. at Eli Lilly, where he works as a senior director and biologist. He and his peers at other companies spent several days learning about these alternatives, discussing the possibilities for their respective companies and seeing the crabs in action. His beach day, he says, was “gratifying.”

“A lot of the time, people don’t think beyond the four walls of the lab,” he says. “But to make that connection, to see it and hear it and smell it, is really cool.”

Harlan, Burgenson and Bolden share a deep love for the species and its natural ecosystem. Although they approach the issue from different worlds—conservation, biotechnology and pharmaceuticals—they all say the new, crabless alternatives are the future. But that future has long faced regulatory roadblocks, conflicting research and an extremely cautious pharmaceutical industry.

Horseshoe crabs predate dinosaurs. Can they survive us?

Larry Niles still remembers a time when it was “unimaginable” that anything could happen to the horseshoe crab population, because the animals were so abundant. As a species, the creatures have been on Earth 1,500 times longer than Homo sapiens. Surely, people thought, we couldn’t do that much damage.

Now, Niles is co-director of the Horseshoe Crab Recovery Coalition, an alliance of 50-plus member organizations that use science, policy and public communication to protect the species from endangerment. The coalition includes Harlan’s organization, the Center for Biological Diversity, which does advocacy for horseshoe crab conservation across industries.

Most areas don’t have specific population counts, but Niles says the American horseshoe crab along the East Coast has seen a decline due to biomedical bleeding and the use of crabs as bait in the fishing industry. The species is described as vulnerable by the International Union for Conservation of Nature. Three other horseshoe crab species exist today, dwelling off the coasts of South Asia and Southeast Asia, where fewer fishing and bleeding regulations exist. One of those three species is designated as endangered. (The others are considered data deficient.)

To create the highly desired product that can test for toxins in medicine—called Limulus amoebocyte lysate, or LAL—fishermen catch horseshoe crabs and bring them to bleeding facilities. There, scientists drain up to 30 percent of each crab’s blood before returning them to the water.

Drawing crabs’ blood isn’t necessarily lethal, and several studies have found an approximately 15 percent mortality rate in the bleeding process. But Niles’ camp believes that even if crabs don’t die, the process can impact their breeding patterns and abilities, thus harming their population and the wider ecosystem.

In a 2022 study in the journal Aquatic Conservation, Niles and others found horseshoe crab egg populations were one-tenth as dense on Delaware Bay beaches than they were 30 years ago, pointing toward the pharmaceutical and fishing industries as the cause. Conservationists emphasize that this decline can affect more than just the crabs: Migrating shorebirds rely on the crab eggs as food during their journey. One such bird, the red knot, was declared threatened in 2015—yet the industries kept using the crabs that fed them.

“Everybody’s after their individual part, and nobody cares about the whole,” Niles says.

Other research, however, has reached different conclusions about the state of this ecosystem. The Atlantic States Marine Fisheries Commission (ASMFC) works with states that bleed horseshoe crabs to track the population status and bleeding impacts across the coast. Using a number of independent surveys, ASMFC reports a steady increase since 2012 in the number of adult female horseshoe crabs in Delaware Bay, which are larger than males and lay eggs during the mass spawnings. This makes females more valuable in the eyes of conservationists, fishermen and biomedical bleeders, and the harvest of female crabs for bait has been prohibited in Delaware Bay for more than a decade. ASMFC’s most recent estimate, in 2022, suggests the bay is home to around 16 million females and 40 million males.

Caitlin Starks, fishery management plan coordinator at ASMFC, says biomedical bleeding has minimal impact on the population as a whole. In the 2022 and 2023 assessments, ASMFC reported that bleeding-related mortality accounted for around 20 percent of total horseshoe crab deaths. The potential negative impacts that don’t result in death are a “data hole,” she says, but ASMFC has seen consistent or improving populations across the coasts in recent years.

“What we’re focused on is making sure that whatever the biomedical industry is doing is sustainable for the horseshoe crab populations,” she says. “And as far as we can tell from our stock assessment, it is.”

Several Plymouth State University studies in the past decade, not connected to Niles and the Horseshoe Crab Recovery Coalition, have mimicked the bleeding process and found horseshoe crabs released after bleeding were about half as likely to come ashore to breed in the following months as those that hadn’t been bled, according to Chris Chabot, a biologist at Plymouth State who worked on the research.

Companies that run bleeding facilities and produce LAL say the methodologies used in the Plymouth State bleeding studies were not reflective of bleeding’s true impacts. Burgenson works at Lonza, one such company, but spoke for this article in his capacity as an adviser to ASMFC and veteran of the biotechnology field. The Plymouth State studies stored crabs in uninsulated barrels for days to simulate two nights at a bleeding facility, but Burgenson says standard industry practice is to catch, bleed and release crabs within 24 hours, closely regulating their temperature while they’re in captivity.

Six companies bleed horseshoe crabs in the U.S., says Starks, and each reports their data and practices to ASMFC. Once they’ve bled the crabs and created the endotoxin tests, these companies sell the testing product to dozens of pharmaceutical companies, which use the tests to confirm the safety of millions of vaccines and medicines.

A line of defense against toxins

Endotoxins, which are passively shed by certain bacteria, are all around us—on our hands and phones, as well as in our mouths and guts. But they can cause fever, organ failure or death if they enter our bloodstreams. Horseshoe crabs’ blood contains a unique enzyme—known as Factor C—that causes clotting as soon as it detects bacterial endotoxins.

In the 1960s, scientists discovered that through this clotting, the enzyme in crab blood could also alert drug manufacturers to endotoxins present in injected medicines, such as vaccines and IV fluids. They developed LAL from horseshoe crab blood to screen for these contaminants, and ever since the product received approval from the U.S. Food and Drug Administration in 1977, companies all over the world have used it in safety testing.

“Years ago, we used to risk our lives to get an injection,” Burgenson says. “Now, nobody thinks twice about going to the pharmacy and getting a flu shot.”

Before horseshoe crab blood, scientists would give lab rabbits a dose of the medicine being tested, then wait to see if they developed a fever. Now, pharmaceutical companies put a small dose of the medicine into the LAL product, and any clotting signals the presence of endotoxins.

Using the crabs’ blood has made endotoxin tests enormously more effective, and demand for the product is only growing as pharmaceutical companies create new drugs. According to ASMFC, the number of crabs bled for biomedical use more than doubled between 2018 and 2023.

With demand already so high, Harlan suggests reliance on horseshoe crab blood could present a supply chain concern if the need for it were to suddenly surge.

“If there is another pandemic, which requires a lot of vaccines, we don’t have the blood supply on hand for the safety tests of all those billions of vaccines that might be required,” he says.

Enter recombinant alternatives

The newly approved alternatives to horseshoe crab blood clone its endotoxin-detecting protein and insert it into insect cells, which are then trained to behave like horseshoe crab cells. Researchers at the University of Singapore created the first genetically engineered alternative to the enzyme in the crabs’ blood in 1997, when they were faced with a lack of horseshoe crabs near them.

The product, called recombinant Factor C, turns fluorescent when it binds with a dangerous endotoxin in a medication. A series of updated versions has been introduced since.

Building on that discovery, Associates of Cape Cod (ACC), a biotechnology company that produces LAL, introduced the first recombinant cascade reagent product in 2021, which cloned not only one protein in horseshoe crab blood, but all relevant pieces. Though this method also announces the presence of endotoxins through changing color, the ACC says the product offers an easier user experience. In an emailed statement, they note that it requires no “specialized equipment [or] specialized training and can be used with existing processes and software.”

Different companies, academics and organizations have conducted dozens of studies on the efficacy of recombinant alternatives compared to the blood-based LAL product. Bolden, of Eli Lilly, says the methods are equivalent, and several studies have shown recombinant alternatives produce fewer false positive results for endotoxins than the blood-based method does. He led a literature review in 2020, published in the PDA Journal of Pharmaceutical Science and Technology, that examined more than ten studies and concluded the two types of products were comparable.

Elizabeth Baker, director of research policy at the Physicians Committee for Responsible Medicine, says these studies get at the heart of the issue—more so than questions surrounding the status of crab populations.

“What I’m going to hang my hat on is that this is better science, and it doesn’t hurt any animals. So, if you’re asking the questions of, ‘Is [the crab population] vulnerable? Is it not vulnerable?’ those are a little less important to me than ‘Is it good science?’” Baker says. She helped organize Bolden’s trip to Delaware Bay last year to educate industry leaders about the possibilities of recombinant alternatives.

Although the alternatives are performing “as well, if not better,” than LAL, they require further testing to verify their safety and efficacy, ACC representatives say in an email. But Burgenson suggests the products will get there.

“I always like to take animals out of the equation, so I see the recombinant assays as eventually supplanting the naturally derived LAL as the products are developed further,” he says.

Charles River Laboratories is the largest producer of the blood-based product, and conservationists like Niles and Harlan cite it as a longstanding hurdle in moving the recombinant alternatives forward. Studies released by the company have questioned their efficacy. But in 2024, the company began offering a recombinant product, and in July, it released a statement highlighting the sustainability of these alternatives and encouraging their use following the updated regulatory guidelines.

“Patient safety” is Charles River’s guiding principle for this issue, says Nora Blair, a senior manager at the company, in an email.

“We refer to a ‘dual-method future,’ because both LAL and recombinant methods have important roles,” Blair says. “LAL has a five-decade track record of patient safety and remains the validated method for many licensed products. Recombinant methods are expanding, but not every product or process has an optimal recombinant test available today.”

Harlan and the Center for Biological Diversity have worked against Charles River to demonstrate the efficacy of recombinant alternatives and make them available. He says this public shift from the company shows that they’re “hedging their bets.”

“What we’re seeing from Charles River is not surprising and reflects what’s happening more broadly across the industry,” Harlan says. “Everyone sees that synthetics are the future.”

Hurdles stand in the way of full approval

No matter where they stand, people on nearly all sides of the issue call the pharmaceutical industry one thing: “conservative.” Both the corporate and government bodies involved in decision-making move at a “glacial” pace, Burgenson says.

For the past two decades, he has been in conversations and research spaces dedicated to moving the crabless alternatives forward. And time and time again, the mechanisms failed to receive regulatory approval. Companies were hesitant to try techniques that required extra hurdles and were not widely used.

“[Pharmaceutical companies] want bigger, better, faster, more,” Burgenson says. “And then when you give them bigger, better, faster, more, they ask, ‘Who else is using it?’”

But between 2016 and 2018, new recombinant products rolled out, and Eli Lilly, with Bolden leading the charge, committed to using these alternatives for testing all new products. In 2020, Europe’s Pharmacopeia approved the alternatives as an equivalent method to LAL. And the U.S. Pharmacopeia voted last year to recognize recombinant alternatives as an option for pharmaceutical companies, a new regulation that took effect this May.

Still, it remains difficult to start testing a legacy medical product with the recombinant alternatives, if it had previously been tested with LAL. The FDA has yet to issue blanket approval for pharmaceutical companies to register new products with the crab-free safety tests, so the companies must demonstrate the suitability of their endotoxin testing plan for each individual product.

But Bolden says he does not anticipate hindrances or extra validation required for registering new drugs with recombinant alternatives. He believes the industry “will see a lot of momentum” around these crabless tests now that fewer policy hurdles remain.

Eli Lilly now tests 80 percent of its products with recombinant alternatives, and companies such as GSK, Amgen and Bristol Myers Squibb have reported that they will begin some sort of transition to the new mechanism. But the wider industry is a little more stagnant—of the 50 largest pharmaceutical companies surveyed by the conservation groups, 39 provided no response about their efforts to shift to these alternatives.

Since the recombinant alternatives were created in the 1990s, this conversation has seen two millennia, five American presidents and the Covid-19 pandemic—and the different perspectives have evolved, shifted and conflicted. But Harlan, of the Center for Biological Diversity, believes that with the introduction of recombinant alternatives into the Pharmacopeia, these moving pieces may finally be aligning.

“We’re a tree-hugging, conservation nonprofit, and we’re working alongside Lilly and the pharmaceutical industry,” Harlan says. “It’s unusual allies, but we’re all in this together, because we all want to see what’s best for the horseshoe crabs, for human health and safety, and for ecosystems.”