Market Alternatives to Wild Harvest
A synthetic alternative to Horseshoe Crab Blood
in Biomedical Testing
Academic & Industry Experts:
Jay Bolden (Eli Lilly and Company), Ling Ding Jeak (National University of Singapore),
Ned Mozier (Pfizer), Larry Niles (Consulting Biologist), and Eric Stiles (New Jersey Audubon).
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 alone, 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 of bled crabs annually sold as bait in other fisheries. The released crabs often suffer sublethal effects of the bleeding process, such as injury and disorientation, increased incidence of disease, and unclear long-term effects possibly including lower spawning rates.
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, despite a decade of 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.
The current overexploitation of horseshoe crabs is dangerously similar to that of other mismanaged species that have been driven to extinction. The International Union for the Conservation of Nature declared in 2019 that one of the three Asian species of horseshoe crab is now endangered. Three years earlier, in 2016, IUCN assessed the mid-Atlantic populations of the American horseshoe crab as vulnerable to extinction 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 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.
Developing the 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 enzyme 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).
Multiple manufacturers subsequently developed their own products. Germany-based Hyglos developed its own version of rFC in 2013, and more recently, Japan-based Seikagaku developed a synthetic that mimics the “full cascade” of enzyme reactions that comprise the LAL test. This product can also replace horseshoe crab blood for a different set of bacterial endotoxin tests. In the United States, the Lonza Group owns the patent for recombinant factor C, which it sells under its own label.
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.
This review paper, “Saving the horseshoe crab: A synthetic alternative to horseshoe crab blood for endotoxin detection,” establishes a path forward for the pharmaceutical industry to eliminate the practice of bleeding horseshoe crabs for biomedical testing. The review paper is the basis of a broad stakeholder engagement effort to shine a light on the opportunity for industry to convert to rFC and contribute to the recovery and sustainability of the horseshoe crab and the birds that depend on them. Revive & Restore’s effort has galvanized a renewed sense of purpose on this issue within the conservation community.
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 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 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.
The existence of an effective synthetic alternative to the LAL test provides the biomedical and pharmaceutical industries the opportunity to modernize procedures and to significantly contribute to the conservation of horseshoe crabs and Delaware Bay shorebirds. In the late 1970s, the pharmaceutical industry transitioned from using live rabbits to detect fever-inducing contaminants to using crabs, now the industry to modernize its methods and use modern technology instead of the blood of an ancient species. 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.
Thanks to the leadership of Eli Lilly and Company, as well as the recently published PLOS review paper, there is now a clear path forward for the pharmaceutical industry to begin converting to the synthetic alternative.
Recombinant factor C has begun getting traction in the market with Eli Lilly’s adoption of the synthetic alternative at several of its U.S. manufacturing facilities. Yet widespread uptake by the industry has been slow, despite the fact that the biomedical industry can switch immediately to the testing of water and other common manufacturing materials with virtually no regulatory oversight. Innovation leadership from within the pharmaceutical industry is needed to improve the sustainability of their manufacturing processes.
Continued technological innovation is also needed. Charles River Labs has developed a micro-fluidic device that reduces the use of horseshoe crab-derived LAL by as much as 80 percent. Until regulators fully adopt rFC as an equivalent test, there will still be demand for LAL. More widespread innovation is needed to limit the use of this product from wild caught sources.
Lastly, given the trend toward rapid product development in the pharmaceutical industry, new companies offering new variants of the synthetic alternative should be entering the market.
Innovation of a synthetic bait alternative may help mitigate the harvest of 500,000 crabs in the bait fishery. However, given the dire resource management constraints of the American eel and whelk fisheries, it is hard to rationalize a significant investment. Each species is suffering from its own form of mismanagement, or in the case of whelk, a lack of management.
Conservatism in the industry has slowed the conversion to rFC, but leadership from other manufacturers in the pharmaceutical industry is now essential. 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.
Abate W, Sattar A, Liu J, Conway M, Jackson S. Evaluation of recombinant factor C assay for the detection of divergent lipopolysaccharide structural species and comparison with Limulus amebocyte lysate-based assays and a human monocyte activity assay. J Med Microbiol. 2017 Jul 12;66(7):888–97. pmid:28693666.
Aketagawa J, Miyata T, Ohtsubo , Nakamura T, Morita T, Hayashida H, et al. Primary structure of limulus anticoagulant anti-lipopolysaccharide factor. J Biol Chem. 1986 Jun 5;261(16):7357–65. pmid:3711091.
Anderson RL, Watson WH, Chabot CC. Sublethal behavioral and physiological effects of the biomedical bleeding process on the American horseshoe crab, Limulus polyphemus. Biol Bull. 2013 Dec;225(3):137–51. pmid:24445440.
Atkinson PW, Baker AJ, Bennett KA, Clark NA, Clark JA, Cole KB, et al. Rates of mass gain and energy deposition in red knot on their final spring staging site is both time- and condition-dependent. J Appl Ecol. 2007 May 9;44(4):885–95.
Baker AJ, González PM, Piersma T, Niles LJ, Nascimento I, Atkinson PW. Rapid population decline in red knots: fitness consequences of decreased refueling rates and late arrival to Delaware Bay. Proc Biol Sci. 2004 Apr 22;271(1541):875–82. pmid:15255108.
Bolden J, Knight M, Stockman S, Omokoko B. Results of a harmonized endotoxin recovery study protocol evaluation by 14 BioPhorum Operations Group (BPOG) member companies. Biologicals. 2017 May;48:74–81. pmid:28549938.
Bolden J, Smith K. Application of recombinant factor C reagent for the detection of bacterial endotoxins in pharmaceutical products. PDA J Pharm Sci Technol. 2017 Sep-Oct;71(5):405–12. pmid:28733334.
Chen L, Mozier N. Comparison of Limulus amebocyte lysate test methods for endotoxin measurement in protein solutions. J Pharm Biomed Anal. 2013 Jun;80:180–5.
Cooper JF, Levin J, Wagner HN. Quantitative comparison of in vitro and in vivo methods for the detection of endotoxin. J Lab Clin Med. 1971 Jul;78(1):138–48. pmid:4936365.
Ding JL, Ho B. A new era in pyrogen testing. Trends Biotechnol. 2001, Aug;19(8):277–81. pmid:11451451.
Ding JL, Ho B, inventors. National University of Singapore, assignee. Assays for Endotoxin. United States patent US 6645724. [Internet] Nov 11 1997. https://patentimages.storage.googleapis.com/c1/74/24/e32003d50429f4/US6645724.pdf
Ding JL, Navas MA 3rd, Ho B. Molecular cloning and sequence analysis of factor C cDNA from the Singapore horseshoe crab, Carcinoscorpius rotundicauda. Mol Mar Biol Biotechnol [Internet]. 1995 Mar [cited 2018 Apr 3];4(1):90–103.
Duijns S, Niles LJ, Dey A, Aubry Y, Friis C, Koch S, et al. Body condition explains migratory performance of a long-distance migrant. Proc Biol Sci. 2017 Nov 15;284(1866). pmid:29093218.
Endangered and threatened wildlife and plants; threatened species status for the Rufa Red Knot. Fed Regist [regulation on the Internet]. 2014 Dec 11;79(238):73706–48. Available from: https://www.gpo.gov/fdsys/pkg/FR-2014-12-11/pdf/2014-28338.pdf.
Grallert H, Leopoldseder S, Schuett M, Kurze P, Buchberger B. EndoLISA®: a novel and reliable method for endotoxin detection. Nature Methods 2011 Sep;8(10).
Iwanaga S. The limulus clotting reaction. Curr Opin Immunol. 1993 Feb;5(1):74–82. pmid:8452677.
James-Pirri M-J, Veillette PA, Leschen AS. Selected hemolymph constituents of captive, biomedically bled, and wild caught adult female American horseshoe crabs (Limulus polyphemus). Mar Freshwater Behav Physiol. 2012 Oct;45(4):281–9.
Levin J, Bang FB. A description of cellular coagulation in the Limulus. Bull. Johns Hopkins Hosp. 1964 Oct; 115:337–45. pmid:14217224.
Levin J, Bang FB. Clottable protein in Limulus; its localization and kinetics of its coagulation by endotoxin. Thromb Diath Haemorrh. 1968 Mar 31;19(1):186–97. pmid:5690028.
Licensing of Limulus amebocyte lysate. Use as an alternative for rabbit pyrogen test. Fed Regist [regulation on the Internet]. 1977 Nov 4;42(213):57749–50.
Loverock B, Simon B, Burgenson A, Baines A. A recombinant factor C procedure for the detection of gram-negative bacterial endotoxin. Pharmacopeial Forum [Internet]. 2010 Jan-Feb [cited 2018 Apr 3];36(1):321–9. Available from: http://www.usppf.com/pf/pub/data/v361/GEN_STIMULI_361_s200140.xml.
McKenzie JH, Alwis KU, Sordillo JE, Kalluri KS, Milton DK. Evaluation of lot-to-lot repeatability and effect of assay media choice in the recombinant factor C assay. J Environ Monit. 2011 Jun;13(6):1739–45. pmid:21552635.
Miyata T, Hiranaga M, Umezu M, and Iwanaga S. Amino acid sequence of the coagulogen from Limulus polyphemus hemocytes. J Biol Chem [Internet]. 1984 Jul 25 [cited 2018 Apr 3];259:8924–8933.
Muta T, Miyata T, Misumi Y, Tokunaga F, Nakamura T, Toh Y, et al. Limulus factor C: an endotoxin-sensitive serine protease zymogen with a mosaic structure of complement-like, epidermal growth factor-like, and lectin-like domains. J Biol Chem [Internet]. 1991 Apr 5;266(10)6554–61.
Nakamura T, Horiuchi T, Morita T, Iwanaga S. Purification and properties of intracellular clotting factor, factor B, from horseshoe crab (Tachypleus tridentatus) hemocytes. J Biochem. 1986 Mar;99(3):847–57. pmid:3519594.
Nakamura T, Morita T, Iwanaga S. Lipopolysaccharide-sensitive serine-protease zymogen (factor C) found in Limulus hemocytes. Eur J Biochem. 1986 Feb;154(3):511–21. pmid:3512266.
Reich J, Heed K, Grallert H. Detection of naturally occurring bacterial endotoxins in water samples. European Pharmaceutical Review [Internet]. 2014 Dec 23;19(6):67–8.
Schwarz H, Schmittner M, Duschl A, Horejs-Joeck J. Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells. PLoS ONE. 2014 9(12):e113840. pmid:25478795.
Smith DR, Beekey MA, Brockmann HJ, King TL, Millard MJ, Zaldívar-Rae JA. Limulus polyphemus. The IUCN Red List of Threatened Species. 2016:eT11987A80159830.
Sweka JA, Klopfer M, Millard M, Olszewski S, Smith D, Sysak R, et al. 2013 Horseshoe crab stock assessment update [Internet]. Atlantic States Marine Fisheries Commission. 2013 Aug.
Thorne PS, Perry SS, Saito R, O’Shaughnessy PT, Mehaffy J, Metwali N, et al. Evaluation of the Limulus amebocyte lysate and recombinant factor C assays for assessment of airborne endotoxin. Appl Environ Microbiol. 2010 Aug;76(15):4988–95. pmid:20525858.
Tokunaga F, Nakajima H, Iwanaga S. Further studies on lipopolysaccharide-sensitive serine protease zymogen (Factor C): its isolation from Limulus polyphemus hemocytes and identification as an intracellular zymogen activated by alpha-chymotrypsin, not by trypsin. J. Biochem. 1991 Jan;109(1): 150–7. pmid:2016264.
Walls EA, Berkson J, Smith SA. The horseshoe crab, Limulus polyphemus: 200 million years of existence, 100 years of study. Rev Fish Sci. 2002;10(1):39–73.
2017 U.S. Pharmacopoeia-National Formulary [USP 40 NF 35] Volume 1. Rockville, Md: United States Pharmacopeial Convention, Inc; 2016.  Bacterial Endotoxins Test. p. 163.