HORSESHOE CRABS IN PERIL
Population threatened by widespread use of crustacean blood in testing, researchers say
Few organisms are as odd, or as old, as the horseshoe crab.
That they predate the dinosaurs might explain their oversized, helmet-shaped shells, which can grow as large as 51 centimetres. Anatomically, they're more like spiders than crustaceans, and they fluoresce under ultraviolet light.
But perhaps their unique feature is how their blood, which is bright blue, coagulates when exposed to harmful bacterial endotoxins, a feature that has kept them alive for about 450 million years.
Bacterial endotoxins induce inflammation and fever, and can cause anaphylactic shock and death. They are responsible for venereal disease, bacterial meningitis, cholera, bubonic plague and other diseases. Immune cells in the crab's blood trap and immobilize these type of endotoxins, rendering them inert.
It's a blessing and a curse because once scientists discovered this amazing defence system back in the 1960s, we began using it for ourselves, bleeding horseshoe crabs and separating out that clotting feature to test medications, needles and biomedical devices to make sure they are contaminant free.
Their blood has been so useful — enabling scientists to create vaccines that help humans fend off everything from migraines to melanomas and most recently the coronavirus — that we've made a dent in their population.
A new technology is available that uses a human-made version of crab blood to detect endotoxins, but it has stirred a debate over whether it is as good. The debate has pitted conservation-minded scientists against a board that sets scientific standards for the pharmaceutical industry, which said more study needs to be done before a synthetic version of crab blood can be used.
Last summer, as coronavirus infection rates continued to rise, a group of researchers from Eli Lilly, Bristol Myers Squibb, Pfizer and Roche-Genentech published a research report that compared the two products — limulus amebocyte lysate, or LAL, which is made from horseshoe crab blood, and the synthetic product, called recombinant Factor C assay, or rFC.
But the findings did little to quell a debate.
Pharmaceutical companies
need to make sure their injectable drugs and medical devices such as hip and knee replacements are free of bacterial endotoxins. But conservationists say rFC can detect bacterial endotoxins equally well. If drug companies continue to rely solely on LAL, they say, horseshoe crab populations will be put at risk.
In the 1990s, the population of horseshoe crabs along the east coast was decimated by fishermen who used them as bait to catch eel and corner the lucrative market for whelk or conch. And the population struggles to rebound as companies that manufacture LAL now harvest crabs. About 500,000 crabs are plucked from waters and beaches along the Atlantic coast each year to make LAL.
“I could show you a movie from 1986 that was filmed right over there, at Reeds Beach, and the eggs were this deep on the beach,” said Larry Niles, a biologist formerly with the New Jersey's Fish and Wildlife division, holding his hand about 20 cm, above the sand.
The eggs are food for migratory shore birds such as the Rufa red knot. But its population fell 75 per cent from the 1980s to the 2000s, in large part because the supply of horseshoe crab eggs dropped.
The hunt for coronavirus vaccines has only fired up concerns about the sustainability of horseshoe crabs. In March 2020, there were 241 therapies, including vaccines, in development. Today, there are about 838, according to Bio, a trade association representing biotechnology companies.
“Every drug or vaccine candidate or clinical trial or finished
solution injected into the body has to have LAL testing. The water and raw materials going into such solutions also have to be tested,” said Kevin Williams, a scientist who spent 30 years at Eli Lilly and now works for bioMerieux, a French multinational biotechnology firm that manufactures rFC.
Bleeding labs, which bleed horseshoe crabs of about 30 per cent of their blood and turn that blood into LAL, collected 637,029 horseshoe crabs in 2019, 30 per cent more than they took the year before.
While the crabs are returned to the water, fishing authorities take it for granted that at least 15 per cent — or 95,554 — of them die. Some research puts that mortality figure as high as 30 per cent.
“As it is now, the entire supply chain for endotoxin testing of drugs rests upon the harvest of a vulnerable or near extinct sea creature,” Williams said. “As prudent as the pharmaceutical industry is, this seems to be a current blind spot.”
To date, Eli Lilly is the only company that uses rFC when submitting its new drug applications to the FDA, although last summer, French drugmaker Sanofi said it, too, planned on using rFC. The European Pharmacopeia approved rFC for endotoxin testing last year.
“While rFC is an alternative to LAL, the data available today is not enough to put them at the same level so they can be used interchangeably,” said Fouad Atouf, vice-president of global biologics at USP.
Atouf said USP isn't the only organization that said this. When it considered putting rFC and
LAL on equal footing in its compendium of standards, it asked industry stakeholders for comment and there was not a broad consensus that there was enough data. Even the FDA expressed concerns, he said.
Jay Bolden, of Eli Lilly, said his study from September provides the USP with that data.
“We looked at all the available scientific literature on rFC, and we found a dozen studies that say exactly what we think the (USP) would need to make those kind of judgments,” Bolden said.
He said there is only one study, from a company that manufactures LAL, in which rFC appears to be inferior, and it is because it used pre-filtered water, which he said can skew the results. Charles River Laboratories International, which did that study, notes it was peer-reviewed, and used samples from various points in the pharmaceutical water purification processes.
Jack Levin, professor at the University of California School of Medicine at San Francisco, and the scientist who helped discover the LAL test, disputes the notion the LAL manufacturers are killing off crabs.
While he acknowledges the crab population in the United States crashed about 15 years ago on account of the bait fishermen, the federal government intervened and instituted quotas, and the population has rebounded, he said.
“People have often approached this with a certain religious fervour and want to ignore that,” he said. “I certainly don't believe in killing animals unnecessarily. And you can argue, if you want to, against animal research, until it impacts your own health, of course. But the argument that the lysate industry is depopulating the crab population is just not correct.”
The American horseshoe crab is not considered endangered, although it's classified as vulnerable on the Red List of Threatened Species of the International Union for Conservation of Nature (IUCN). But conservationists fear if it continues to be overfished, the species could go the way of the Asian horseshoe crab, which is already extinct in Taiwan, disappearing in Hong Kong and dwindling in China. The IUCN lists the Asian crab as endangered because of biomedical bleeding, as well as coastal development destroying its habitat.
The USP said it is committed to finding ways to transition from animal-derived materials to synthetic ones, but it needs more proof the two products are on par. The USP is conducting a large study of its own this summer comparing the two products, though much to the dismay of those pushing for rFC, the study will also use some water samples that are unfiltered, not unlike the water used in the Charles River study.
Jessica Ponder, a regulatory testing analyst for the Physicians Committee for Responsible Medicine, said the American crab population may not be on the brink right now, but we don't want to be reliant on an animal, particularly one that can be found in only one country now. Her organization, which promotes in vitro assaid to replace animal testing where they're not necessary, said she has looked at the test and believes the data to use rFC is there.
There's a reason Eli Lilly has switched over to rFC, and it's not just good stewardship but foresight, she said.
“They see this coming a mile away, that eventually we're not going to have this horseshoe crab blood available,” she said. “Is that going to be today? Is it going to be 20 years from now? That's not something that's easy to predict when you have a natural resource, but at the same time, what are we waiting for?”
As our reliance on these crabs grows, they continue to do their job, emerging from the sea each May when the tide is high and the moon is full, and climbing up onto the beaches to spawn. For now, they are saving us. One day, it may be the other way around.