“Horseshoe crabs have blue blood. Instead of iron, their blood contains
copper, manifesting in the striking color. Photos: Timothy Fadek/Redux”
SYNTHETIC HORSESHOE CRAB BLOOD
https://audubon.org/biomedical-revolution-to-save-horseshoe-crabs
https://students.bowdoin.edu/synthetic-horseshoe-crab-blood
https://theconversation.com/synthetic-horseshoe-crab-blood
New synthetic alternatives mean pharma won’t bleed this species dry
by Kristoffer Whitney & Jolie Crunelle / October 12, 2023
“If you have ever gotten a vaccine or received an intravenous drug and did not come down with a potentially life-threatening fever, you can thank a horseshoe crab (Limulus polyphemus). How can animals that are often called living fossils, because they have barely changed over millions of years, be so important in modern medicine? Horseshoe crab blood is used to produce a substance called limulus amebocyte lysate, or LAL, which scientists use to test for toxic substances called endotoxins in intravenous drugs.
These toxins, produced by bacteria, are ubiquitous in the environment and can’t be removed simply through sterilization. They can cause a reaction historically referred to as “injection fever.” A strong concentration can lead to shock and even death. Identifying LAL as a highly sensitive detector of endotoxins was a 20th-century medical safety breakthrough. Now, however, critics are raising questions about environmental impacts and the process for reviewing and approving synthetic alternatives to horseshoe crab blood. We study science, technology and public policy, and recently published a white paper examining social, political and economic issues associated with using horseshoe crabs to produce LAL. We see this issue as a test case for complicated problems that cut across multiple agencies and require attention to both nature and human health.
Doctors began injecting patients with various solutions in the mid-1800s, but it was not until the 1920s that biochemist Florence Seibert discovered that febrile reactions were due to contaminated water in these solutions. She created a method for detecting and removing the substances that caused this reaction, and it became the medical standard in the 1940s. Known as the rabbit pyrogen test, it required scientists to inject intravenous drugs into rabbits, then monitor the animals. A feverish rabbit meant that a batch of drugs was contaminated. The LAL method was discovered by accident.
Working with horseshoe crabs at the Marine Biological Laboratory at Woods Hole, Massachusetts, in the 1950s and ’60s, pathobiologist Frederik Bang and medical researcher Jack Levin noticed that the animals’ blue blood coagulated in a curious manner. Through a series of experiments, they isolated endotoxin as the coagulant and devised a method for extracting LAL from the blood. This compound would gel or clot nearly instantaneously in the presence of fever-inducing toxins. Academic researchers, biomedical companies and the U.S. Food and Drug Administration refined LAL production and measured it against the rabbit test. By the 1990s, LAL was the FDA-approved method for testing medicines for endotoxin, largely replacing rabbits.
“Rabbits undergoing a pyrogen test in a laboratory
in 1956 to determine a drug’s safety”
Producing LAL requires harvesting horseshoe crabs from oceans and beaches, draining up to 30% of their blood in a laboratory and returning the live crabs to the ocean. There’s dispute about how many crabs die in the process – estimates range from a few percent to 30% or more – and about possible harmful effects on survivors. Today there are five FDA-licensed LAL producers along the U.S. East Coast. The amount of LAL they produce, and its sales value, are proprietary. As biomedical LAL production ramped up in the 1990s, so did harvesting horseshoe crabs to use as bait for other species, particularly eel and whelk for foreign seafood markets. Over the past 25 years, hundreds of thousands – and in the early years, millions – of horseshoe crabs have been harvested each year for these purposes. Combined, the two fisheries kill over half a million horseshoe crabs every year.
There’s no agreed total population estimate for Limulus, but the most recent federal assessment of horseshoe crab fisheries found the population was neither strongly growing nor declining. Conservationists are worried, and not just about the crabs. Millions of shorebirds migrate along the Atlantic coast, and many stop in spring, when horseshoe crabs spawn on mid-Atlantic beaches, to feed on the crabs’ eggs. Particularly for red knots – a species that can migrate up to 9,000 miles between the tip of South America and the Canadian Arctic – gorging on horseshoe crab eggs provides a critical energy-rich boost on their grueling journey. Red knots were listed as threatened under the Endangered Species Act in 2015, largely because horseshoe crab fishing threatened this key food source. As biomedical crab harvests came to equal or surpass bait harvests, conservation groups began calling on the LAL industry to find new sources.
Many important medicines are derived from living organisms. Penicillin, the first important antibiotic, was originally produced from molds. Other medicines currently in use come from sources including cows, pigs, chickens and fish. The ocean is a promising source for such products. When possible, synthesizing these substances in laboratories – especially widely used medications like insulin – offers many benefits. It’s typically cheaper and more efficient, and it avoids putting species at risk, as well as addressing concerns some patients have about using animal-derived medical products.
“A sample of horseshoe crab blood Florida Fish & Wildlife Commission“
In the 1990s, researchers at the National University of Singapore invented and patented the first process for creating a synthetic, endotoxin-detecting compound using horseshoe crab DNA and recombinant DNA technology. The result, dubbed recombinant Factor C (rFC), mimicked the first step in the three-part cascade reaction that occurs when LAL is exposed to endotoxin. Later, several biomedical firms produced their own versions of rFC and compounds called recombinant cascade reagents (rCRs), which reproduce the entire LAL reaction without using horseshoe crab blood. Yet, today, LAL remains the dominant technology for detecting endotoxins in medicine.
The main reason is that the U.S. Pharmacopeia, a quasi-regulatory organization that sets safety standards for medical products, considers rFC and rCR as “alternative” methods for detecting endotoxins, so they require case-by-case validation for use – a potentially lengthy and expensive process. The FDA generally defers to the U.S. Pharmacopeia. A few large pharmaceutical companies with deep pockets have committed to switching from LAL to rFC. But most drug producers are sticking with the tried-and-true method. Conservation groups want the U.S. Pharmacopeia to fully certify rFC for use in industry with no extra testing or validation. In their view, LAL producers are stalling rFC and rCR approval to protect their market in endotoxin detection. The U.S. Pharmacopeia and LAL producers counter that they are doing due diligence to protect public health.
Change may be coming. All major LAL producers now have their own recombinant products – a tacit acknowledgment that markets and regulations are moving toward Limulus-free ways to test for endotoxins. Atlantic fisheries regulators are currently considering new harvest limits for horseshoe crabs, and the U.S. Pharmacopeia is weighing guidance on recombinant alternatives to LAL. Public comments will be solicited over the winter of 2024, followed by U.S. Pharmacopeia and FDA review. Even if rFC and rCR don’t win immediate approval, we believe that collecting more complete data on horseshoe crab populations and requiring more transparency from the LAL industry on how it handles the crabs would represent progress. So would directing medical companies to use recombinant products for testing during the manufacturing process, while saving LAL solely for final product testing. Making policy on complex scientific issues across diverse agencies is never easy. But in our view, incremental actions that protect both human health and the environment could be important steps forward.”
“Horseshoe crabs are bled at the Charles River Lab in Charleston, SC”
BLEEDING HORSESHOE CRABS
https://nytimes.com/drug-safety-horsehoe-crab
https://phillymag.com/horseshoe-crab-orgy-season
https://theguardian.com/horseshoe-crabs-overharvesting
Horseshoe crabs under threat from overharvesting
by Robin McKie / 2 Mar 2024
“They are some of the most spectacular images currently on display at the Wildlife Photographer of the Year exhibition in London. Huge pictures of weird creatures with golden carapaces and multiple legs scuttling across the seabed now adorn the walls of the city’s Natural History Museum. The award-winning photographs, taken by Laurent Ballesta, could be visions of extraterrestrial lifeforms. In fact, they are horseshoe crabs that evolved hundreds of millions of years ago and which are some of Earth’s oldest species. These living fossils thrive on the ocean floor where they root out worms, algae, and clams that they crush between their legs before eating. The biological relationship between horseshoe crabs and Homo sapiens is distant.
Yet these strange creatures have become a vitally important cog in modern life – for their bright blue blood is a critical component of tests to assess the safety of vaccines, insulin injections and many other medical interventions used by humans today. “Harvesting horseshoe crabs for their blood has saved millions of lives,” said Rich Gorman of Sussex University, author of a recent study of the horseshoe crab. “If you’ve ever been given a vaccine, you can thank the horseshoe crab for ensuring that your jab was safe.” But the exploitation of the horseshoe – which is more closely related to spiders than crabs – has come at a price, added Gorman. Biologists say they are now under grim evolutionary pressures that are triggering precipitous declines in their numbers, particularly on the eastern seaboard of America. Hundreds of thousands are having their blood taken, triggering swathes of deaths and reducing populations in key breeding areas such as Delaware Bay in New Jersey.
This decrease has also had wider ecological consequences. Major drops in populations of birds, such as the rufa red knot, are now being reported in the wake of the horseshoe’s decline. Every spring, thousands of red knots used to fly to Delaware Bay from the tip of South America and gorge themselves on the millions of eggs laid there by crabs during the horseshoe spawning seasons. Then the birds would fly off for the remainder of their 9,000-mile migration to the Arctic. However, recent drastic drops in red knot numbers have been reported, and these have, in turn, been blamed on the decline of the horseshoe crab. In addition, horseshoes provide fishermen with a key source of bait, and this widespread exploitation has also contributed to the ecological pressures on the horseshoe.
“Horseshoe crabs on Moores Beach / Photograph by Doug Wechsler”
As a result, a ban was imposed this year on the harvesting of female horseshoes in Delaware Bay. But many ecologists believe that there is no longer an excuse for using horseshoes, either for bait or as a source of medical materials. “The fisheries – such as eel fishing – that used horseshoe crabs as bait are dwindling, and there are alternatives to using their blood for medical products,” said Larry Niles, a wildlife biologist in New Jersey. “So there’s no reason to be killing these animals at all.”
“Blue blood being extracted from horseshoe crabs before they
are returned to the shoreline. Photograph: Ariane Mueller”
This last point is backed by scientists. They say that viable alternatives to toxicity tests using horseshoe blue blood have been on the market for 15 years. These rely on synthetic ingredients and if implemented across the globe could lead to the end of horseshoe bleeding. Only a few organisations – such as Eli Lilly – have chosen to adopt the new synthetic test, mainly because there was no regulatory encouragement for companies to swap techniques.
However, recent changes in the rules set by medical regulators, who set quality and identity standards for medicines, have raised hopes that tests based on horseshoe medicine bleeds could be discouraged and replaced with those that use synthetic ingredients. European, Japanese, and Chinese pharmacopoeias have passed regulations aimed at securing such a goal, and a similar decision is expected in the US in the near future. “Changing the rules will certainly help but we will still have to persuade companies that they need to change their practices and save the horseshoe crab,” said Niles. “They won’t do that overnight.”
Horseshoe crabs evolved around 450 million years ago, long before the appearance of dinosaurs. There are four species: three from Asia and one that lives along the east coast of North America. The last is the main source of crabs for bleeding. The blood of horseshoes contains cells that are exceptionally sensitive to toxic bacteria. When those cells meet invading micro-organisms, they form clots and so protect the rest of the horseshoe crab’s body, a phenomenon that scientists exploited to develop the limulus amebocyte lysate (LAL) test in the 1970s. This can determine if medical products, such as vaccines, have been contaminated with dangerous bacteria.
To obtain supplies of the blood needed to make these tests, hundreds of thousands of horseshoe crabs are gathered every year. Their blood is extracted and the animals are then returned to their shoreline homes. One estimate, made in 2021, suggested that of 718,809 horseshoe crabs collected for these bleedings, a total of 112,104 died, around 15%. Other conservation groups put that death toll at around 30% or more, however. “About half of a crab’s blood is taken from each animal and that is simply too much,” said Larry Niles, a wildlife biologist in New Jersey. “They are being killed in vast numbers, and that is having severe ecological consequences not just for the crabs but for other creatures that eat the eggs that the horseshoe crab lays.”