Written by Kristine Yang '25
Edited by Lizzy Zhang '24
Horseshoe crabs have existed for nearly 450 million years, predating even the earliest of dinosaurs [1]. Yet despite their age, these living fossils continue to provide one of the most expensive liquids on the market: their bright blue blood.
This horseshoe crab blood – blue due to a copper-based pigment – is the only known natural source of limulus amebocyte lysate (LAL), a substance that can detect even the smallest traces of a dangerous bacterial contaminant called endotoxin. Upon entry into the human bloodstream, endotoxin can be deadly [2].
Thus, since its discovery in 1956, LAL tests have been used almost universally by biotechnology and pharmaceutical companies in quality and safety screening for medicines, injectable drugs, and implantable devices such as insulin, vaccines, and artificial knees and hips. Recently, the expedited creation and production of millions of safe COVID-19 vaccines largely relied on LAL tests [3].
These critical benefits to human health are nonetheless reliant on the crude LAL extraction process in which horseshoe crabs are captured and bled before being returned to the ocean. With rates of mortality before their return to the ocean increasing due to the capturing and bleeding process, the ever-growing utilization and industry reliance on LAL test are rapidly driving the depletion of wild horseshoe crab populations [4].
In response to growing concerns over the sustainability of using LAL tests and the uncertain future supply of horseshoe crab blood, biotechnology and pharmaceutical companies have been exploring new alternatives for endotoxin screening.
One alternative predates the universal adoption of the LAL test. Before the abilities of horseshoe crab blood was discovered, the industry standard for endotoxin screening was called the United States Pharmacopeia (USP) rabbit pyrogen test that involved measuring the temperature change of live rabbits following the injection of the tested product [4]. The test produced qualitative results with low sensitivity, and incited concerns over testing variability and the obvious ethical issue with using live animals [5].
Forward-looking researchers and companies are in favor of exploring synthetic alternatives for endotoxin screening.
One test that has gained traction is the recombinant factor C (rFC) test which makes use of a cloned reagent extracted from the DNA of a horseshoe crab, and like the LAL test, triggers clotting when in the presence of endotoxins [4]. Not only does the rFC test eliminate the need for wild horseshoe crab capture, it also has been shown to be more sensitive than the LAL test due to having multiple potential endotoxin binding sites [4]. In 2018, the FDA approved the first drug that used an rFC reagent test to screen for endotoxins for safety control during its production, opening the door for wider adoption of this testing method [6].
Another synthetic alternative to the LAL test is the Monocyte Activation Test (MAT) that uses the innate human immune system response and completely eliminates the need for any horseshoe crab byproduct. This test uses monocytes, a type of white blood cells in humans, and measures their response to contaminants and toxins [4]. However, this test lacks the sensitivity needed to detect endotoxin on medical surfaces and can take up to 20 hours to process, therefore reducing its practicality and appeal as a replacement for LAL tests [4].
Though both synthetic alternatives, rFC and MAT, methods produce similar results to the prominent LAL test while also conserving wild horseshoe crab populations, the adoption and replacement of LAL tests by these alternatives remain a challenge due to a reluctant and hesitant industry [4]. The switch to these newer endotoxin screening methods would require complex validation and the redesigning of manufacturing processes that have been in place for more than 40 years.
References
[1] Smithsonian’s National Zoo, Conservation Biology Institute. 10 incredible horseshoe crab facts [Internet]. Smithsonian’s National Zoo & Conservation Biology Institute. 2022 [cited 2022 Dec 13]. Available from: https://nationalzoo.si.edu/animals/news/10-incredible-horseshoe-crab-facts
[2] Arnold C. Horseshoe crab blood is key to making a COVID-19 vaccine—but the ecosystem may suffer. National geographic [Internet]. 2020 Jul 2 [cited 2022 Dec 13]; Available from: https://www.nationalgeographic.com/animals/article/covid-vaccine-needs-horseshoe-crab-blood
[3] Pavid K. Horseshoe crab blood: the miracle vaccine ingredient that’s saved millions of lives [Internet]. Nhm.ac.uk. [cited 2022 Dec 13]. Available from: https://www.nhm.ac.uk/discover/horseshoe-crab-blood-miracle-vaccine-ingredient.html
[4] Krisfalusi-Gannon J, Ali W, Dellinger K, Robertson L, Brady TE, Goddard MKM, et al. The role of horseshoe crabs in the biomedical industry and recent trends impacting species sustainability. Front Mar Sci [Internet]. 2018;5. Available from: http://dx.doi.org/10.3389/fmars.2018.00185
[5] Sigmaaldrich [Internet]. Sigmaaldrich.com. [cited 2022 Dec 13]. Available from: https://www.sigmaaldrich.com/US/en/applications/microbiological-testing/pyrogen-testing
[6] USP provides guidelines for Recombinant Factor C (rFC) a non-animal-derived reagent critical to development of vaccines and other sterile pharmaceutical products [Internet]. Usp.org. [cited 2022 Dec 13]. Available from: https://www.usp.org/news/rfc-horseshoe-crabs-statement
[IMAGE] Ho S. Near threatened horseshoe crab blood remains standard for big pharma drug testing [Internet]. Green Queen. 2020 [cited 2022 Dec 13]. Available from: https://www.greenqueen.com.hk/near-threatened-horseshoe-crab-blood-remains-standard-for-big-pharma-drug-testing/