Crabby concoctions: why pharma’s thirst for crustacean blood is up for debate
Horseshoe crabs' icy-blue blood is the drug industry's standard for safety tests. Despite efforts to increase the use of a synthetic substitute, regulators have decided that the fake stuff doesn’t quite cut the rug compared to the real thing. Abi Millar finds out what makes this blue-blooded crustacean so appealing to drug developers.
For the past four decades, the pharma industry has found an unlikely ally in the form of the horseshoe crab. This ancient species – which, at 450 million years old, is sometimes called a ‘living fossil’ – has a unique immunological response to bacteria. This means its blood, which is rich in copper and icy blue in appearance, can be used to detect certain contaminants in vaccines, infusions and implanted medical devices.
“The animal’s so-called immunity does not involve production of antibodies, like humans,” explains Dr Anthony Dellinger, president of Kepley Biosystems. “Rather, it releases proteins that have the ability to recognise invasive bacteria in parts per trillion. It forms an immediate gel clot around the bacteria, trapping and killing the pathogen.”
While the animal is not killed for its blood, there are many questions surrounding its welfare. An estimated 130,000 die during the harvesting or bleeding process every year, and those that survive are returned to the ocean many miles from where they were originally captured.
“If they do make it back, they’re in a new environment, and they’ve lost a third of their blood,” says Ryan Phelan, co-founder and executive director of the conservation non-profit Revive & Restore. “On top of that, the horseshoe crab is considered a keystone species, because many other species depend on it. Every year, it releases millions of eggs along the coastline on the high tide, and migratory birds come from all over the world to feed on those eggs.”
The upshot is that harvesting has become a major problem, with their numbers dwindling and the surrounding ecosystems in jeopardy. A debate has been raging for some time: does the industry really need to use the limulus amebocyte lysate (LAL) that it derives from the crabs’ blood? Or should they adopt a synthetic equivalent?
The synthetic substitute
For groups such as Revive & Restore, the question is a no-brainer. Recombinant factor C (rFC), which can be manufactured in a lab, has been available since 2003. The pharma giant Eli Lilly is already using rFC in place of LAL and has released two therapies that were safety tested this way. What’s more, the European, Japanese and Chinese pharmacopoeias all recognise rFC as an equivalent.
“Eli Lilly had a really strong corporate philosophy about removing the use of an animal product,” says Phelan. “And quite honestly, what they were able to show was that rFC wasn’t just equal to LAL – it was better. They did have to retool their desktop and use a different kind of testing process, and there’s a cost involved in that. But it’s just a de minimis amount of money compared to drug discovery.”
In 2018, Revive & Restore published a review article that synthesised ten studies on rFC. It found that rFC is just as efficacious and safe as LAL, that it’s more consistent and cost effective, and that switching to rFC would result in a 90% drop in demand for horseshoe crab blood.
What they were able to show was that rFC wasn’t just equal to LAL – it was better.
However, the US Pharmacopeia (USP), which issues quality standards for these tests, is yet to put the two tests on an even footing. Initially, the organisation had proposed adding rFC to its existing chapter governing endotoxin testing standards.
But on 29 May it made a surprise U-turn, announcing the synthetic test would be placed in a standalone chapter. Drug companies wishing to use rFC will need to do extra work to validate their methods.
USP representatives said since they have 30 years of drug manufacturing data on LAL, and only two years on rFC, they needed more safety information.
“There’s actually 15 years of clinical data on rFC, so when they reversed their proposal, we were all blown away,” says Phelan.
“They said it was because of stakeholder feedback. So, Revive & Restore submitted a request for disclosure, and we got back copies of the letters that were submitted in comment. The two most negative, disconcerting letters are not surprisingly from companies that do the bleeding – two companies with total vested interest in maintaining the market for LAL.”
The aquaculture option
Dellinger has a different take on the situation. He believes that, rather than switching to rFC, the industry needs to move towards horseshoe crab aquaculture and produce the LAL more sustainably. His company, Kepley Biosystems, has developed a model for doing so. Its research suggests that horseshoe crab husbandry and bleeding is achievable without ill effect to the animal.
“Our work involves robust species knowledge and scientifically derived husbanding and bleeding knowhow,” he says. “Animals should be cared for thoughtfully, bleeding should be low-impact and controlled, and conservation efforts should be multifaceted.
"The animals move, exercise, socialise and express normal reproductive behaviours. Our accomplishments are remarkable because 100% of our animals are alive and well, vibrant and lively.”
He believes that rFC is no substitute for LAL, and adds that the USP panel members are all noted academics who considered the analogue fairly (“if this is not true, then the entire world of pharmaceutical manufacturing and public health is in serious trouble”, he remarks).
The argument here boils down to the idea that, as a natural substance that has evolved over millions of years, LAL has some complexities that are hard to replicate. Unless you fully understand its mechanisms of action, you’d struggle to create a precise analogue.
Bleeding should be low-impact and controlled, and conservation efforts should be multifaceted.
“The LAL cascade is a handshake of specialised enzymatic reactions that work in concert to perform the task of gram-negative bacteria detection,” says Dellinger. “That is to say, Factor C is one component in this opera, but we should not discount the roles of Factor B, preclotting enzymes and coagulogen, and how each of these can contribute to the substrate’s performance.”
His company believes LAL testing will soon emerge as the gold standard in early diagnosis of infectious diseases. As well as endotoxin testing in manufacturing, it could be used across a number of other translational innovations.
“The horseshoe crab has a lot to teach research scientists,” he says. “As an ancient species that has remain morphologically unchanged for more than 200 million years, their biology can hold a plethora of information in an age of antimicrobial resistance and viral pandemics.”
An ongoing debate
Phelan isn’t convinced. While she wouldn’t want to dismiss anyone’s entrepreneurialism, she feels a movement towards horseshoe crab aquaculture is misguided.
“Why would you continue to use a wild species when you don't have to?” she says. “If there were no synthetic equivalent, and the horseshoe crab blood helped to actually save human lives, you bet I’d be thinking about the healthy way to harvest these animals.
“But there’s no reason to do it. We have a biotech equivalent and it’s better. The idea that you’re going to take this species that takes ten years to become reproductively viable and keep them in captivity... it boggles the mind.”
I think the industry is going to move forward using the new European standard, rather than waiting for the USP.
Revive & Restore is currently leading the charge to demand the USP reverse its decision. However, Phelan doesn’t think the industry is going to wait around.
“I think the industry is going to move forward using the new European standard, rather than waiting for the USP,” she says. “There’s growing public awareness around the horseshoe crab and the environmental issues involved in that. So, any manufacturer can plan to utilise the European Pharmacopeia standard and put their products forward.”
Given the current need for new vaccines – and associated demand for endotoxin testing – we can expect this debate to gain momentum. Existing methods of LAL production are not sustainable – that much we know for sure. But time will tell whether the industry moves towards a synthetic equivalent, or simply works to improve its treatment of this unlucky blue-blooded crustacean.