Ocean Genomics Horizon Scan

Synthetic Alternatives: Horseshoe Crabs

Pictured Above: The Atlantic Horseshoe Crab, Limulus polyphemus – the only extant species of horseshoe crab native to the Americas.

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 the horseshoe crab was developed several decades ago, but market adoption has been minimal, primarily because of lingering doubts over its efficacy.

Experts Interviewed

Jay Bolden, Eli Lilly and Company
Ling Ding Jeak, National University of Singapore
Ned Mozier, Pfizer
Eric Stiles, New Jersey Audubon
Larry Niles, Consulting Biologist

Pictured Above: Horseshoe crabs are bled at the Charles River Laboratory in Charleston, South Carolina. Photograph: Timothy Fadek.

Why the Pharmaceutical Industry Depends on the Horseshoe Crab:

Four species of 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. 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’s 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 – every shot, IV drip, or implanted medical device – 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 of 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. Although some estimates are higher, at least 15 percent die from the bleeding procedure, with a similar percentage sold as bait in other fisheries.

The Horseshoe Crab as Bait

In addition to the bleeding for LAL, there is a fishery for horseshoe crab, which in the US, is used as bait in the whelk and eel fisheries. The Atlantic States Fisheries Commission now regulates the number of crabs harvested for bait, after overharvesting in the 1990s caused a population crash. Today, with conservation management, the horseshoe crab population in Delaware Bay (the largest population in the U.S.) is still depleted to 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 is ecologically unsustainable.

Ecosystem-Wide Impacts of Horseshoe Crab Harvesting

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 spring migration along the Atlantic flyway to gorge on the eggs of spawning horseshoe crabs in Delaware Bay, a critical food stop on their journey north to Arctic nesting grounds, 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 (Pictured Below) 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. 


Pictured Above: Ling Ding Deak, co-inventor of recombinant Factor C.

Development of a Synthetic Alternative

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). 

Demonstrating Efficacy

More than a decade of research has proven that for the detection of gram-negative bacterial endotoxin, rFC 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. However, the pharmaceutical industry has been slow to adopt rFC due to concerns over efficacy, among other issues.

The California conservation nonprofit Revive & Restore set out to dispel lingering doubts over efficacy of the rFC synthetic alternative. The results, which were published in PLOS Biology in 2018, demonstrated that a synthetically produced test for bacterial contaminants was just as effective as the LAL test produced with blood components of the horseshoe crab. Revive & Restore analyzed data from ten separate and independent studies, each showing that efficacy and reliability of the synthetic alternative was equal to or better than the product derived from horseshoe crab blood.

“Saving the horseshoe crab:
A synthetic alternative to horseshoe crab blood for endotoxin detection.” 


Adoption by the Pharmaceutical Industry 

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, a step that alone reduces the number of LAL tests performed at each facility by 90 percent, according to endotoxin experts with decades of experience. The company took an even bigger step forward in its adoption of the synthetic alternative this fall, 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.

Immediate conversion to rFC for the testing of water and other common manufacturing materials presents no risk of diminution in reliability or sensitivity in endotoxin detection and 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, which means that mortality resulting from bleeding would decrease.

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, sport fisheries, and the estuaries of the Eastern Seaboard.

Without any change to the current drug manufacturing regulations in the United States, pharmaceutical companies can significantly reduce their unsustainable dependence on a wildlife product by converting to the equally safe and effective synthetic alternative for testing only common manufacturing materials.

To fully ensure the adoption of the synthetic alternative, rFC needs to be validated as an acceptable endotoxin detection method by regulatory bodies around the world. Because vaccines and drugs are manufactured and distributed worldwide, various regulatory bodies (e.g., FDA) rely on different compendia (e.g., US Pharmacopeia) and, where possible, a harmonization process to assure uniformity in endotoxin testing methods across all regulatory jurisdictions. Because the use of rFC detection methods has not been incorporated as an accepted method into the harmonized Pharmacopeias, for final-product testing manufacturers must take the extra step of validating the rFC assay, which is a more burdensome process than the streamlined method of verification used for accepted methods described in the harmonized Pharmacopoeias.


Recent scientific advancements only build upon decades of conservation efforts for both crabs and shorebirds spearheaded by conservation groups including National Audubon, New Jersey Audubon, the American Littoral Society, and the Western Hemisphere Shorebird Reserve Network, as well as government agencies like U.S. Fish & Wildlife Service. 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 studied the relationship between migratory shorebirds and spawning horseshoe crabs; and advocated for the adoption of the synthetic alternative to LAL.