Development Of Synthetic Endotoxin Detection Method Could Protect Horseshoe Crabs From Harm During Biomedical Testing

Four species of the horseshoe crab, the Atlantic horseshoe crab (Limulus polyphemus) and three additional Asian species, have been integral to the safe manufacturing of injectable medications for the past 40 years. Populations of all four extant species of horseshoe crab are in decline across the globe, in part because of the extensive use of horseshoe crab blood in biomedical testing. The bleeding of live horseshoe crabs, a process that leaves thousands dead annually, is an ecologically unsustainable practice for not only the horseshoe crab but also the shorebirds that rely on their eggs as a primary food source during spring migration. A synthetic alternative to the biomedical test derived from horseshoe crab was developed several decades ago, but market adoption has been minimal, primarily because of lingering doubts over its efficacy.

The California conservation nonprofit Revive & Restore set out to dispel these doubts, analyzing ten independent studies that each demonstrated the efficacy and reliability of this synthetic alternative. The resulting review paper, ‚ÄúSaving the horseshoe crab: A synthetic alternative to horseshoe crab blood for endotoxin detection,‚ÄĚ was published in PLOS Biology in 2018 and establishes a path forward for the pharmaceutical industry to eliminate the practice of bleeding horseshoe crabs for biomedical testing.


Horseshoe crab blood is extraordinarily sensitive to bacterial contaminants that can cause life-threatening fever and toxic shock if introduced to the bloodstream. A unique clotting protein in the crab‚Äôs blood is extracted to create the Limulus amebocyte lysate (LAL) test, which is used to screen every injectable drug approved by the U.S. Food and Drug Administration. In fact, anything that might go inside the human body ‚Äď every shot, IV drip, or implanted medical device ‚Äď is tested with LAL for contamination.

An estimated 70 million LAL tests are performed each year, which, in the United States, results in the bleeding around 500,000 horseshoe crabs annually. The horseshoe crabs are captured and drained of as much as a third of their blood before being returned to the ocean. An estimated 15 percent die from the bleeding procedure, with a similar percentage sold as bait in other fisheries. Released crabs often suffer sublethal effects of the bleeding process, such as injury and disorientation, increased incidence of disease, and possibly lower spawning rates.

Today, the horseshoe crab population in Delaware Bay (the largest population in the U.S.) is depleted from its use as bait for the welk and eel fisheries; the population is now estimated at about a third of the Bay’s carrying capacity. It is from this already depleted population that crabs are harvested for bait and for use in the pharmaceutical industry. The slow recovery of horseshoe crabs makes it clear that the current levels of harvest for bait and LAL manufacturing are ecologically unsustainable.

The current overexploitation of horseshoe crabs is dangerously similar to that of other mismanaged species that have been driven to extinction. In 2016, the International Union for the Conservation of Nature moved the mid-Atlantic populations one step closer to extinction on its Red List. Furthermore, in the mid-Atlantic region of North America, the overharvest of the horseshoe crab is causing significant ecosystem-level impacts. The six species of shorebirds that synchronize their northward migration along the Atlantic flyway to gorge on the eggs of spawning horseshoe crabs in Delaware Bay are some of the most rapidly declining shorebirds in North America. In 2014, the dwindling horseshoe crab population in North America prompted the classification of the red knot (Calidris canutus rufa), whose 9,500-mile migration from the tip of South America to the Arctic is among the longest of any bird in the world, as threatened under the US Endangered Species Act.


But the pharmaceutical industry now has an extraordinary opportunity to help conserve the 450 million-year-old horseshoe crab and, in so doing, transform the conservation of migratory shorebirds, sports fisheries, and the estuaries of the Eastern Seaboard.

In 1997, scientists Ling Ding Jeak and Bow Ho of the University of Singapore developed a synthetic version of factor C, the key molecule in horseshoe crab blood that coagulates in the presence of endotoxins. This reaction is the basis of the LAL test. This synthetic alternative, produced using recombinant DNA, is known as Recombinant Factor C (rFC). While the pharmaceutical industry has been slow to adopt rFC, more than a decade of research has proven that for the detection of gram-negative bacterial endotoxin, the synthetic alternative is just as effective as the LAL assay, if not more effective, in its ability to quantifiably measure endotoxin and in its ability to detect endotoxins across a range of concentrations.

Immediate conversion to rFC to test the water and other common manufacturing materials presents no risk of diminution in reliability or sensitivity in endotoxin detection, and the process is enabled under current regulatory guidance. Most importantly, converting to rFC for only the testing of common pharmaceutical manufacturing materials like water would decrease the demand for LAL by 90 percent, according to endotoxin experts at pharmaceutical manufacturer Eli Lilly and Company, with decades of experience.

Eli Lilly has become the first pharmaceutical manufacturer to adopt rFC. Three of its largest U.S. manufacturing facilities are now testing pharmaceutical water and other common manufacturing materials using the synthetic alternative. The company took an even bigger step forward in its adoption of the synthetic alternative in 2018 when its migraine prevention drug (galcanezumab), that had been tested only using the rFC assay, was approved by the FDA. Eli Lilly’s progressive pivot towards rFC is the result of years of research on the efficacy of the synthetic alternative by one of the company’s top biologists. Leadership from other manufacturers in the pharmaceutical industry is now essential.

Recent scientific advancements only build upon decades of conservation efforts for both crabs and shorebirds spearheaded by Delaware Bay regional conservation groups like New Jersey Audubon, the American Littoral Society, and the Western Hemisphere Shorebird Reserve Network, as well as national agencies like U.S. Fish & Wildlife Services. These groups have pushed the Atlantic States Fishery Commission to include the biomedical take of horseshoe crabs in its regional annual fishery catch limits, extensively studying the relationship between migratory shorebirds and spawning horseshoe crabs and advocating for the adoption of the synthetic alternative to LAL.


These findings are described in the article entitled Saving the horseshoe crab: A synthetic alternative to horseshoe crab blood for endotoxin detection, recently published in the journal PLOS Biology.



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