Turning tides for endotoxin testing

At a recent meeting of the Parenteral Drug Association, where industry microbiologists discussed ways to make drugs without a trace of unwanted biological material, photos of horseshoe crabs danced across a screen between sessions. The sediment-snuffling arthropod with a dozen legs and a shell like a helmet may seem like an unlikely pairing with the sleek, highly engineered robotics of a pharmaceutical production line. But estuaries teeming with life and clean rooms where it should be all but absent are linked by their dependence on this animal.

That is poised to change. In November, US regulators will formally announce their acceptance of alternatives to a key test that ensures drug products are not contaminated. The new tests will use proteins produced in bioreactors rather than in wild horseshoe crabs.

The change was hard fought. Arguments played out in the technical literature and in expert committee meetings. The stakes included hundreds of millions of dollars in annual sales for the company with the most to lose from a switch and the professional reputations of a group of industry insiders who were fired from their volunteer jobs. Now it appears that the tide may be turning on endotoxin tests made from horseshoe crab blood—but it hasn’t gone out just yet.

Limulus lysate

“Every human who has medical intervention has benefited from horseshoe crabs,” says Glenn Gauvry, president of a horseshoe crab–focused nonprofit called the Ecological Research and Development Group. Any injected drug or implanted device is tested for endotoxins, which are signature glycolipids from the outer membranes of certain bacteria and can harm people even if no viable bacteria are present. Horseshoe crab blood contains a sensitive biosensor system that pharmaceutical companies use to detect those glycolipids.

And it’s not used to test only products that go into humans. Veterinary medicines, water and raw materials, drug containers, media used to grow cells that produce biologics—all these and more are tested routinely. According to the endotoxin test vendor bioMérieux, market research suggests that 70 million–100 million endotoxin tests are conducted globally each year. That number is forecast to grow along with growth in demand for biological drugs, which require especially extensive testing.

According to Thomas Hartung, who directs the Center for Alternatives to Animal Testing at Johns Hopkins Bloomberg School of Public Health, endotoxin testing is worth between $500 million and $1.5 billion in the US annually. “It is a very big business,” he says, and companies in the space are “aggressive” about protecting their interests.

The Atlantic States Marine Fisheries Commission estimates that about 700,000 crabs are collected in the US each year for biomedical use. Manufacturers disinfect the crabs and drain some of their hemolymph—the crab equivalent of blood, which happens to be blue. They then isolate and lyse blood cells, producing a reagent called limulus amoebocyte lysate, or LAL.

Though the crabs are later released, scientists estimate that between 4 and 30% later die. Environmentalists say that survivors may not be healthy enough to spawn—putting pressure on migratory birds that depend on the crabs’ bountiful eggs.

Decades ago, Jeak Ling Ding and her colleagues at the National University of Singapore cloned the protein in horseshoe crab blood cells that senses lipopolysaccharides (Mol. Mar. Biol. Biotechnol. 1995, 4, 90). Endotoxin assays based on that protein, known as recombinant factor C (rFC), have been sold by the company Lonza since 2003 and by bioMérieux since 2016.

Jay Bolden, an avid birder and a senior director at the pharmaceutical company Eli Lilly and Company, has been working since 2013 to adopt the recombinant tests at Lilly. In 2018, the US Food and Drug Administration approved the company’s first fully rFC-tested drug, Emgality, a monoclonal antibody used to prevent migraines. After that, many scientists expected that broader adoption of rFC-based tests would come quickly. A cover story in the Atlantic proclaimed that “the last days of the blue-blood harvest” had arrived.

Yet 6 years later, uptake remains limited. That’s because pharmaceutical companies have to do a lot more work if they want to use rFC—not because the tests themselves are more complicated but because regulatory requirements consider LAL the gold standard.

David Smith, a vice president at BioCentriq, a contract development manufacturing organization focused on cell therapies, says his company has not yet adopted recombinant endotoxin tests because to use them, manufacturers in the US must submit an extra dossier of experiments to the FDA. That would add thousands of dollars to the cost of each contract for BioCentriq’s customers.

The US Pharmacopeia’s role

The authority to change requirements for drug quality assurance tests lies with national pharmacopoeias. These expert bodies produce protocols for making medicines safely, testing them for contaminants, and packaging them appropriately.

The European Pharmacopoeia, under pressure from the European Union, began to recognize recombinant endotoxin tests in 2015 as part of a broader move away from animal testing. China, where horseshoe crabs are endangered, adopted the new tests starting in 2020. But unlike these pharmacopoeias, the US Pharmacopeia (USP) is not part of the government.

“USP itself is an independent body. It’s a nonprofit organization and not a regulatory one,” says Leslie Furr, current staff liaison to the USP Microbiology Expert Committee, which wrote the new rules being published next month. But, she adds, “the standards that we set are recognized under federal law, and while we are different entities, the USP and FDA do collaborate together on the standards.”

While the FDA had signaled openness to consider recombinant endotoxin tests in 2012, the USP took more time.

“To the consternation of people who use rFC, and to people in the FDA I’ve spoken to, the FDA defers to the USP,” says Kristoffer Whitney, a professor at Rochester Institute of Technology who studies horseshoe crabs and their interactions with human society. Whitney says the USP’s microbiology committee “had for years been sort of the holdup.”

The USP depends on panels of expert volunteers to set its policies. For years, the microbiology committee comprised scientists with long experience in the pharmaceutical industry; many remembered the adoption of LAL back in the 1970s.

Committee members knew that their decisions would be enforced by the FDA and were of great interest to the companies they’d regulate. That interest isn’t unique to endotoxin tests. According to David Hussong, a former microbiology committee chair, when the committee put together a sterile filtration chapter, filter companies scrambled to make sure the wording favored their products. The work took years, he says, “because we were very careful not to favor one organization over another. But they pushed. Boy, did they push.”

What was different when the committee discussed rFC was that beyond vendors, there were also environmentalists campaigning on behalf of horseshoe crabs and the birds that rely on them.

To avoid an appearance of bias, the USP doesn’t adopt tests sold by just one company; rFC became eligible for inclusion only after the second rFC test reached the market, in 2016. In 2019, the USP announced a plan to accept rFC tests as equivalent to LAL and invited stakeholders to comment. For a time, it appeared to outsiders that recombinant endotoxin tests would soon be formally accepted in the US. But within the committee, there was friction, which favored the status quo.

And then the pandemic hit.

Pandemic supply chain concern

As multiple companies rushed to develop vaccines against COVID-19, observers began to worry about whether the horseshoe crab population could accommodate a sudden demand for billions of vaccines.

“There’s always been this big question mark around sustainability,” Smith, the BioCentriq executive, says. “At what point do we exhaust the market for horseshoe crab blood?”

The answer has not been easy to come by. Because the blood is processed into LAL, it’s hard to define a relationship between crabs caught and tests performed, and LAL producers have continually improved their processes to use smaller volumes. But the concern attracted enough attention to earn a 2020 article in the New York Times.

During the pandemic, “we didn’t understand why the media had gotten so deeply involved in this, how they’d been recruited to take positions,” says James Akers, a former member of the microbiology committee and its endotoxin subcommittee.

That interest was the work of campaigners at the conservation group Revive and Restore. Although the campaigners were influencing public opinion, the group’s executive director, Ryan Phelan, says, she felt boxed out of the rooms where decisions were made. “We have a very limited reach in what we can do. I mean, we can get an op-ed in the New York Times, we can get a cover story in the Atlantic, but you know, that only reaches certain people.”

Members of the USP’s microbiology committee bristled when Jaap Venema, the chief science officer of the USP, mentioned during a technical webinar that the organization was examining ways to allow new tests while still guaranteeing vaccine safety in case of a shortage of LAL. Hussong says he saw this statement as managerial overreach into a key job of the committee: to ascertain that the new tests were as safe as what the industry had already been using.

The persistence of doubt

In April 2020, the committee reversed its plan to include recombinant tests in the main chapter for endotoxin testing, citing feedback from the comment period. Instead, the committee proposed publishing an informational chapter about using recombinant tests. Under this plan, drugmakers who wanted to use recombinant tests would still need to do extra validation experiments.

“It caught us all by surprise,” environmentalist Lawrence Niles says.

When the draft informational chapter came out later that year, it revealed a few of the committee’s objections to the new endotoxin tests.

One concern was about production. Whereas the FDA regulates LAL as a blood product, the agency had decided back in 2002 not to regulate recombinant reagents. The committee worried that this lack of regulation could compromise quality. Several speculated that anyone could bottle anything and call it rFC. The committee recommended that buyers of alternative endotoxin tests audit the test manufacturers’ facilities and conduct more extensive validation than for other quality-control tests, like polymerase chain reaction.

Another issue was statistical. To make sure an endotoxin test works properly, technicians always run controls, including a positive control. A technician pipettes a known quantity of validated lipopolysaccharide into a sample of drug product and tests for the molecules they know are there. The committee was concerned about how precisely the measurement should match the expected concentration of lipopolysaccharide. The requirements for LAL tests had been carried over from the days of twofold dilutions, and the committee argued that it was time to tighten up the rigor.

The committee’s third concern was the identity of those spiked-in endotoxins. Lipopolysaccharide is not just one compound; it’s a whole family of molecules. The committee argued that microbial contaminants were likely to come from water systems and that tests should be able to detect real-world contamination. Instead of a reference standard from Escherichia coli, the committee proposed that pharmaceutical companies validating recombinant tests should seek out unpurified water from their own facilities to use as a positive control.

In public comments received late in 2020, scientists from across the industry and from the FDA weighed in on these technical concerns. Many sounded taken aback by the committee’s suggestions, pointing to positive experiences by drug manufacturers who were already working with recombinant tests. The Parenteral Drug Association wrote that the chapter “seems to be attempting to dissuade the use of rFC” by introducing “exclusive hurdles for rFC compared to other USP guidance.”

Commenters also pointed out echoes between the committee’s concerns and those of the leading LAL producer, Charles River Laboratories. In his written submission, David Klug of the drug company Sanofi observed that some of the points in the text seemed similar to those “coming from an internal study from Charles River . . . that is not published in a peer review[ed journal].” Those points seemed designed to scare off potential users of the recombinant assay, Klug wrote.

The campaign

Few companies produce LAL in the US because of regulations on the horseshoe crab fishery and on the production of the test. Charles River is the most prominent. According to the firm’s website, it supplies the endotoxin tests for 55% of global injected and implanted products. The company did not respond to repeated requests for comment on this story.

Every LAL vendor, including Charles River, has developed recombinant alternatives to horseshoe crab blood. In a February 2024 podcast, the company’s now-retired director of research and development for microbiological solutions, Norman Wainwright, said, “It was a long process.”

Meanwhile, the company employed lobbying firms to “work on the need for Limulus Amebocyte Lysate (LAL) product and synthetic products to be regulated similarly in terms of safety and effectiveness” and for “standards for developing synthetic alternatives for bacterial endotoxin testing,” spending at least $470,000 between 2020 and 2023, according to public disclosures accessed through the nonprofit group OpenSecrets.

Charles River eventually published the study that Klug said he encountered, which compared different assays’ detection endotoxins in unpurified manufacturing facility water and concluded that recombinant tests were inferior (Eur. J. Pharm. Sci. 2021, DOI: 10.1016/j​.ejps.2021.105716). When others in the field objected to the study’s design, the journal’s editors investigated. They determined that while the results were valid for the specific case described in the article, the authors generalized too broadly from the types of samples they had tested when they concluded that recombinant tests were inferior (Eur. J. Pharm. Sci. 2021, DOI: 10.1016/j.ejps.2021.105877).

In a petition to the FDA to recognize rFC, bioMérieux describes the water study as “misinformation . . . accompanied by false, full page advertisements that rFC cannot recognize bacterial endotoxins of the very types already demonstrated to be equally recovered” in a past study.

‘The committee kind of dug in its heels’

Throughout this period, the endotoxin subcommittee’s meetings became more contentious. The informational chapter that had garnered skeptical responses from the FDA and various pharmaceutical companies was canceled. But many committee members with concerns about rFC objected to the cancellation and wanted to see more data comparing rFC and LAL head to head.

One person particularly opposed to adopting recombinant tests commissioned baseball hats emblazoned with “Show me the data” for others who agreed. Akers says that while no one wore these hats to meetings, some did share photos of themselves wearing them with the rest of the endotoxin subcommittee.

“We were not saying no,” says Jim Agalloco, another member of the microbiology committee at the time. “We were just saying, ‘Not yet, not so fast.’ But that was not good enough.”

“Members of the committee that weren’t involved in endotoxin were getting frustrated,” Akers says. “Other things that critically needed to be discussed and thought about were back-burnered.”

Then came a last, disastrous meeting of the endotoxin subcommittee in September 2022. According to committee members spoken to by C&EN, the especially skeptical member behind the hats had learned about a meeting between USP staff and endotoxin test vendors—a meeting to which most of the expert committee had not been invited. The committee demanded to know more about the content of this meeting. Members voted to throw out the committee’s planned agenda to focus instead on what one member called “subterfuge” by the pharmacopoeia. Tempers became heated.

Within the week, committee members received notice that the committee was being dissolved. In a notice on its website, the USP wrote, “This action was taken due to fundamental issues with the dynamic of this group that resulted in an inability to work collaboratively and productively, impeding the standards-setting process.” In the letter informing the committee of the decision, the USP’s Venema wrote, “All current committee members who commit to working collaboratively . . . will be welcome to reapply for membership.”

Few did. Many felt stung after years of volunteering. Former subcommittee member Russell Madsen says, “USP wanted to go one direction. The committee kind of dug in its heels and said no, and then we said hell no, and at that point, that was it.”

“Some of the recombinant reagent vendors had suggested that the committee was in bed with the LAL vendors and creating a barrier” to recombinant endotoxin reagents, Hussong says. But he and each of the 5 of 14 committee members who spoke to C&EN for this story denied these allegations. Instead, the committee members C&EN interviewed say they were concerned primarily about patient safety.

New rules

In its initial announcement about dissolving the microbiology committee, the organization said, “USP is furthering innovation, including alternative methods for endotoxin testing, and is ready to collaborate with companies who adopt these methods to collect more real-world evidence, accelerate scientific knowledge and broaden the adoption of animal-free reagents and methods.”

The pharmacopoeia enlisted a new set of volunteers in 2023, including Lilly’s Bolden. The new committee moved fast. Within the year, without much apparent drama, it completed a draft of a new chapter on recombinant endotoxin tests. In July, after assimilating feedback that Furr, the staff liaison, said led to no major changes, the committee voted to approve the chapter. The final text will be published for early adoption on Nov. 1 and is due to take effect in May 2025.

In January 2024, Charles River Laboratories launched its recombinant endotoxin test, making it the last of the LAL suppliers to introduce such a product.

Several commenters noted the coincident timing. “Things only changed when the market leader, Charles River . . . came up with a recombinant version. Suddenly they stopped to fight it. And now it’s going to be accepted,” says Hartung, the director of the animal alternatives center.

According to Michael Reynier, a senior vice president at bioMérieux, the stage is set for pharmaceutical manufacturers in the US to pivot quickly away from testing facility water systems and other parts of the manufacturing process using animal-derived reagents. Companies may also begin to use the new endotoxin tests for new drug filings.

Changes to existing drug products will take more time because changing any part of the quality assurance process requires new filings with regulators. Despite having committed to adopt recombinant tests in 2016, Lilly is just “80% converted,” Bolden says. “It’ll be hard to change that last 20%” because of the complexity of updating regulatory authorization for numerous drugs in 130 countries. Similarly, even though recombinant testing has been compendial in the European Union for years, many companies there still rely on LAL, Whitney, the Rochester Institute of Technology professor, says.

At a microbiology meeting of the Parenteral Drug Association held Oct. 7–9 in Washington, DC, bioMérieux, Associates of Cape Cod, and Charles River were the top three corporate sponsors, and all had stands in the exhibit hall advertising their recombinant reagents. The vendors, along with competitors Lonza and Fujifilm Wako Chemicals, appear poised to fight for market share in the US. The next battle may be between rFC-based tests and those using recombinant cascade reagents.

Meanwhile, Phelan has begun planning the next phase of Revive and Restore’s horseshoe crab campaign: a scorecard that tracks companies’ environmental promises and whether they are winding down their use of LAL.

Concerns about low or slow uptake of the test concern Gauvry, president of the horseshoe crab nonprofit, who recently released a white paper that says the new regulations are no panacea for crab populations threatened by habitat loss and unregulated harvesting in some parts of the world.

Still, Gauvry commends the approval of recombinant tests—especially if drug companies use them widely. And for the pharmaceutical industry veterans who have been fighting for the tests for years, the new regulation is “a generational change,” Lilly’s Bolden says. “It’s a big win.”