Horseshoe crabs shed for their blue blood. That practice will end soon. Horseshoe crabs’ blood is extensively collected to retrieve the important cell for medical research. However, recent innovations can make this practice obsolete. The blue blood of horseshoe crabs is so valuable that a quarter can be sold for $ 15,000. This is because it contains a molecule that is important to the medical research community.
Today, however, new innovations have resulted in a synthetic alternative that could end the breeding of horseshoe crabs for their blood. One of humanity’s strangest and scariest activities is slowly coming to an end, a trend that every horseshoe crab should celebrate. Yet at the moment, hundreds of thousands of horseshoe crabs are being harvested in the sea off the east coast of the US and they are shedding their precious blue blood.
It is a real practice, but there is a good reason for it. Limulus polyphemus, the Atlantic horseshoe crab, contains extremely valuable blood. Unlike the blood of vertebrates, horseshoe crabs do not use hemoglobin to carry oxygen throughout their bodies. Instead, they use hemocyanin, a chemical that gives your blood its distinctive blue color, but this is not what makes your blood so valuable. Instead, it is the type of immune cells they carry.
life saving blue blood
Vertebrates carry white blood cells in their bloodstream; Instead, invertebrates like the horseshoe crab carry amoebocytes. When an amoebocyte comes into contact with a pathogen, it releases a chemical that causes localized blood clotting, which researchers believe is a mechanism for isolating dangerous pathogens.
Specifically, amoebocytes in horseshoe crab blood accumulate when it comes in contact with endotoxin, a widespread and sometimes fatal bacterial product that activates the immune system, sometimes resulting in fever, organ failure, or septic shock.
The presence of endotoxins in drugs, needles, or anything that comes into contact with human blood is a serious problem. The researchers gave the rabbits a sample of whatever material or substance they were interested in and watched them for hours to see if their immune systems were reacting, indicating the presence of endotoxin.
But amoebocytes in horseshoe blood were a game changer: Instead of performing time-consuming tests on rabbits, horseshoe crab amoebocytes could be added to a sample of a substance. If the sample started to clot, there were endotoxins.
The substance obtained from horseshoe blood is called limulus amebocyte lysate, or LAL, and it quickly became as valuable as gold. Thanks to the ubiquity of endotoxins and the dire need to test for their presence, a quarter of horseshoe crab blood can fetch up to $ 15,000. To profit, the companies harvest 600,000 crabs a year.
Up to 30% of their blood is shed before returning to the sea, although this painful process apparently has some mortality. Estimates vary wildly. Some official sources have estimated the mortality rate at around 3-4%, but these figures generally reflect the mortality rate from direct transport and handling. Other organizations put the death rate at 30%.
Fortunately for horseshoe crabs, this practice may be coming to an end. The researchers found that a molecule in LAL called factor C was responsible for its clotting action. The researchers genetically modified the guts of insects, which are similar to the horseshoe crab, Arthropoda, to produce factor C. As a result, the worms start pumping out factor C, which can then be marketed as recombinant factor C (RFC). Market as a viable alternative to horseshoe crab blood.
Although RFC has been on the market since 2003, it has been slow to gain traction. Initially, it was produced by a single manufacturer, the Lonza Group. Pharmaceutical companies are wary of relying on a manufacturer in an emergency, and their supply is cut off. The Food and Drug Administration (FDA) regulatory process was also very slow. But these obstacles are gradually being removed.
Another pharmaceutical manufacturer, Hyglos GmbH, began producing rFCs in 2013. European regulatory bodies have approved its use, which forms the basis for future FDA approval. The major pharmaceutical companies using RFCs have confirmed that it works in the same way as LAL. Today, experts believe that RFCs will become the dominant method of endotoxin detection, scaring off horseshoe crabs.
Last days of the blue blood harvest
Each year more than 400,000 crabs are hunted for the miraculous healing substance that cleanses their bodies; now, drug companies are finally committed to an alternative that does not harm animals.
A gloved hand holding a syringe for the shell of a horseshoe crab
Horseshoe crabs are sometimes called “living fossils” because they have existed in some form for more than 450 million years. In this time, Earth has gone through several major ice ages, a great death, the formation and subsequent disintegration of Pangea, and an asteroid impact that again killed the dinosaurs and most of life on Earth. In other words, the horseshoe crabs have seen some dirt.
Still, I suppose, some of their strangest experiences may have occurred in the last few decades, when dinosaurs, one of the soft-bodied mammals, began using their hands to throw horseshoe crabs into the sea. Contemporary humans do not intentionally kill horseshoe crabs, as farmers in centuries past captured them to use as fertilizer or as bait for fishermen. Instead, they clean the barn crabs, fold their hinged rugs, and drive stainless steel needles into a soft, vulnerable spot to draw blood. Horseshoe crab blood runs blue and opaque like antifreeze mixed with milk.
And what exactly does man need from the blood of a living fossil? A kind of witchcraft, one might say, because it literally keeps people alive. Horseshoe crab blood is very sensitive to toxins from bacteria. It is used to test for contamination during the manufacture of anything that enters the human body – every injection, every IV drip, and every implanted medical device.
The modern biomedical industry relies so much on this blood that the disappearance of horseshoe crabs would immediately paralyze it. And in recent years, horseshoe crabs, especially in Asia, have faced several threats: loss of habitat due to seawalls replacing beaches where they lay eggs, pollution, food and forage, for use as catching more fish. . Blood-soaked horseshoe crabs are returned to the ocean for biomedical use in the United States, but an estimated 50,000 also die in the process each year.
Horseshoe crabs being transported at the Charles River Laboratory in Charleston, South Carolina (Timothy Phadeck / Corbis / Getty)
There is another way, though: a way for modern medicine to use modern technology instead of the blood of an ancient animal. Horseshoe – A synthetic alternative to crab blood has been available for 15 years. It’s a story about how scientists managed to quietly overcome millions of years of evolution and why it took the rest of the world so long to catch up.
Jake Ling Ding says she was “always a lab rat,” the kind of biologist who wore a white coat instead of digging in mud. However, in the mid-1980s, she found herself walking through the mud looking for horseshoe crabs. The estuary where she lived, she recalls her, in fashion, “didn’t smell sweet at all.”
Ding, along with her husband and research partner Bo Ho, came to the loop near the horseshoe crabs, and her ultimate goal was to make the animals no longer essential in biomedical research. At the time, she was a molecular biologist at the National University of Singapore, and a hospital’s in vitro fertilization department had come to Ding and Ho with a problem: Their embryos would not live long, either from bacterial contamination. .can?
A standard test from that era, and now, is LAL, which stands for Limulus Amoebocyte Lysate. Limulus refers to Limulus polyphemus, a species of horseshoe crab native to the Atlantic coast of North America. Amoebocyte refers to the cells in the crab’s blood. And the lysate is the material that is released from cells once they “smooth” or break down. This is extremely sensitive to bacterial toxins.
The human immune system can be much more sophisticated than that of the horseshoe crab, but it also reacts to these toxins. Doctors first realized this in the late 1800s, when patients contracted “injection fever” or “saline fever” despite having received sterile injections. At worst, the toxins can cause septic shock and even death.
When Bang was doing this research in the 1950s, the standard way to test for bacterial toxins was to inject a sample into rabbits. The rabbit’s temperature needs to be checked every 30 minutes for signs of fever, which would suggest bacterial contamination.
Under the microscope, rabbit blood cells also had a tendency to clump around venom, a similarity Bang noted in his 1956 paper on horseshoe crab blood. Over the next decade and a half, he and a young pathologist named Jack Levine came up with a standardized way to eliminate LAL.
However, it wasn’t until 1977 that the Food and Drug Administration allowed drug companies to replace their large rabbit colonies with LAL kits. Now simply add LAL to the tested content and flip the vial to see if it solidifies, much faster and more convenient. LAL testing still required the use of animals, but the horrible process of sticking needles in animals was hidden and outsourced to a different part of the supply chain.
By the time Ding was hunting for horseshoe crabs in Singapore, LAL had become a multi-billion dollar industry. A quarter of horseshoe crab blood reportedly costs $ 15,000. And the LAL kits he needed to test IVF embryos for contamination were too expensive. He recalls that a kit cost him $ 1,000 in Singapore.
So he thought of making his own lysate. But the species of horseshoe crab he was studying in Singapore, Carcinoscorpius rotundicauda, is much smaller than Atlantic horseshoe crabs and cannot shed much blood without dying. So Ding set out to create an alternative to LAL, which would eventually not require horseshoe crabs.
This would require manipulating the DNA. His idea was to split the horseshoe crab gene responsible for LAL’s toxin-hunting ability in cells that grow easily in a laboratory, such as yeast. Biotechnology as a field was already moving toward recombinant DNA, which involves taking DNA from one species and inserting it into another. A few years ago, in 1982, Eli Lilly began selling human insulin grown in bacteria vats.
Ding had a good starting point for his LAL option. By then, scientists had identified factor C, the LAL-specific molecule that detects bacterial toxins. He then began looking for the gene that produces factor C. His research team took cells from horseshoe crabs that they collected and minimally bled. (He also tried growing horseshoe crabs in a lab and raising them via IVF, but was unsuccessful.)
Unfortunately, the horseshoe crab’s sensitivity to bacterial toxins made it a pain to study. It turns out that toxins are everywhere: in water, in test tubes, in Petri dishes. “You have to bake all the bakeable glassware at 200-220 degrees for several hours,” says Ding. They also had to buy special water that turned out to be free of bacterial toxins. If he’s not careful, his tube of solution can easily turn to gel.
When Ding and Ho finally identified the factor C gene, they divided it into yeast. This failed because the yeast produced factor C, but did not secrete the molecule. “It was very difficult to break down the yeast. It was very dirty and messy, “he says. They tried other types of yeast and mammalian cells, but were not successful either.
In the late 1990s, Ding and Ho attended a course in the United States and learned about the baculovirus vector system. Here, a virus is used to inject factor C into the intestinal cells of insects, turning them into tiny factories of the molecule. Insects and horseshoes have a common evolutionary lineage: they are both arthropods. And these cells did wonders.
Horseshoe crabs shed
Finally, a decade and a half after starting, Ding had an alternative to LAL that worked without damaging more horseshoe crabs. He got involved in the library to study the patent and wrote the application himself. He then he sent it out and waited for the world to change. The world hasn’t changed, at least not for horseshoe crabs.
It took three years for the first recombinant factor C test kit to arrive, based on the Ding patent in 2003, but even then there was little interest from drug companies. The companies had many reasons. There was only one supplier of the kit, a company that is now part of the Swiss-based chemicals company Lonza. Pharmaceutical companies were wary of relying on a single source for such a significant part of their manufacturing.
What if something happens to Lonza? Or did a natural disaster affect your production plant? Companies that bleed crabs would end up losing a lot of money if the C factor is widely adopted. Of the six companies with crab bleeding facilities in the United States, two declined interviews, one did not respond to an interview request, and two have virtually no public appearance. The sixth is Lonza, which currently sells both LAL and recombinant factor.
Slow regulation was blamed for the transport of horseshoe crabs to suck blood at the Charles River Laboratory in Lonza, Charleston, South Carolina. In the United States, the FDA requires companies that test for bacterial toxins to comply with the United States Pharmacopeia, a booklet that details drug standards.
In the 2012 guidance, the FDA stated that companies can use recombinant factor C, which is not listed in the pharmacopoeia, if they have performed its validation tests. “Of course, there is a risk that the FDA will not accept your verification and you will not be able to market your product,” says Lonza spokeswoman Katrin Hoek. “Pharmaceutical companies avoid risk.” It also took decades for the industry to go from rabbits to LAL.
The reality of the business turned out to be a real disappointment to Ding. “We were very curious as researchers, and we are glad that it is working,” he says. And we thought that recombinant factor C would be adopted around the world and the horseshoe crab would be saved. Recently, however, little has changed the recent risk-reward calculation for drug companies.
For one thing, Lonza is no longer the sole provider. In 2013, Hygloss became the second company to manufacture recombinant factors. Kevin Williams, a senior scientist at Hygloss, says he sees this as a long-awaited modernization: Pharmaceutical companies stopped relying on pigs and began producing insulin in yeast and bacterial cells for decades. First. Why can’t the same technology be applied to the test used to see if insulin is safe to inject?
On the regulatory side, the European Pharmacopoeia added recombinant factor C as a bacterial toxin test approved in 2016, paving the way for change in the United States. Several pharmaceutical companies, notably Eli Lilly, have compared the efficacy of recombinant factor C and LAL. Jay Bolden, a bacterial toxin detection specialist at Eli Lilly, recalls that Lonza created the recombinant factor C kit in his lab more than a decade ago.
At the time he was curious, but he was not yet ready to take the plunge. The turning point came in 2013, when Eli Lilly began planning an insulin manufacturing plant in China, where the native species of horseshoe crab is declining. “Someday you’ll hear about the horseshoe crab,” says Bolden. In contrast, the supply chain for recombinant factor C appeared to be more secure, as did Hyglos and Lonza’s suppliers. LAL and Factor C are also comparable in cost.
Bolden says Eli Lilly decided to “draw a line in the sand”: After a certain point, all new products will be tested with recombinant factor C. Recently, the company submitted its first application to the FDA for a drug, galcanezumab to prevent migraine, where the final quality test of the drug would be with factor C. It has also focused on the use of recombinant factor C during the manufacturing process. to analyze water and equipment, which currently accounts for the vast majority of LAL use.
Bolden says Eli Lilly is in the US to include recombinant factor C. Defenders of the Pharmacopoeia. On Thursday, Bolden will speak at an event in Cape May, NJ, organized by Revive & Restore, a nonprofit known for its work to resurrect extinct species. “Our mission is to use biotechnology for conservation,” says Ryan Phelan, co-founder and CEO of Revive & Restore. Phelan met Ding, when he went to Singapore for the synthetic biology conference in 2017.
And realized that his research on recombinant factor C is completely at the intersection of conservation and biotechnology. Revive & Restore and its conservation partners, the New Jersey Audubon, the American Littoral Society, and the Delaware River Keeper Network, chose the Cape location because horseshoe crabs flock here every spring. Horseshoe crabs can no longer be caught here due to their importance to a threatened migratory bird species called the red knot.
These birds also appear here in spring. Their migration is timed so that birds flying from South America to the Arctic can gorge themselves on caviar-like horseshoe crab eggs. The beaches turn black with the crabs, their shells clicking as the females rush to lay their eggs and the males to fertilize them. The red bales rush to eat. They weigh almost twice as much for a trip to the Arctic. This is an ancient inter-species synchrony, which began long before humans began collecting horseshoe crabs for blood, and is expected to last for a long time.