By Julia Simpson
Imagine for a moment you’re in a bar with a group of friends; it’s science-themed trivia night – which you’re attending, because you’re awesome – and the announcer says, “okay folks, final question: the modern medical research industry and healthcare system depend on harvesting a critical chemical from what blue-blooded ocean-dwelling creature?”
Thanks to this article, you’ll now be able to carry your team to victory, if this scenario becomes a reality. The answer is the horseshoe crab. You’re welcome.
Horseshoe crab blood cells are called amoebocytes1, and in 1968, scientists discovered that these cells could coagulate, or clot, in the presence of endotoxins – critical components of bacterial cell walls. This ability made amoebocytes invaluable to the medical field because every injectable medicine produced worldwide must be tested for endotoxin contamination2, due to the reality that endotoxins can cause serious inflammatory responses and even toxic shock if introduced into the human bloodstream3. Versions of the horseshoe crab-derived Limulus amoebocyte lysate (LAL) test, where “Limulus” is the scientific name for the horseshoe crab species, quickly became and have since remained the most widespread way to screen for endotoxins2. However, the annual “blood harvest4” is devastating to horseshoe crab populations, which have also been increasingly threatened by overfishing, habitat loss, and pollution5. Horseshoe crabs may carry the very lifeblood of the biomedical industry in their veins, but they are also integral to many coastal ecosystems6. For example, horseshoe crab eggs are a food source for certain species of shorebirds, so their decline has an environmental ripple effect6. The situation begs the question: can we find an alternative way to test for endotoxins that does not require draining the blood from hundreds of thousands of horseshoe crabs each year?
As it so happens, we already have a synthetic alternative: recombinant factor C (rFC). Factor C is the protein in amoebocytes that allows for endotoxin detection, and rFC is a lab-produced molecule made by taking the Factor C gene from amoebocytes and inserting it into the DNA of microorganisms, turning them into microscopic rFC factories7. Furthermore, rFC has proven capable of detecting endotoxins5. Problem solved, right?
Well, not quite. rFC has been commercially available since 20035, and yet after almost two decades, the blood-harvest-dependent LAL test remains the dominant method for screening endotoxins. In the years since the development of the rFC test, numerous research efforts have investigated the efficacy of the rFC test in comparison to the existing LAL test. Notably, several major studies, recounted in Maloney et al.’s comprehensive 2018 review article3, have found that
a) rFC is the more specific of the two tests and is not susceptible to returning false positive results, while LAL is8;
b) rFC can detect endotoxins as reliably as LAL in a variety of different water9 and air samples10; and
c) rFC has a high sensitivity11 to a broad range of bacterial endotoxin molecular structures3.
These last points are important because scientists need to know that rFC tests are as reliable at detecting endotoxins across the same vast range of circumstances as LAL tests are. Taken together, these data provide strong evidence that rFC represents an innovative, ecologically conscious alternative to LAL endotoxin testing. However, despite the years’ worth of such evidence accumulated, the pharmaceutical industry has been unwilling to make the switch to rFC over LAL on any meaningful scale.
One reason for their hesitation is caution. The clear danger of endotoxins, and the existence of a known reliable test for their detection, has deterred companies from feeling the need to switch over from the LAL test they already trust. Another major reason that pharmaceutical companies have been reluctant to switch to rFC is the system of stringent regulatory processes, dictated by various agencies and organizations worldwide, that regulate acceptable methods of endotoxin testing during drug development. The rFC endotoxin test was deemed acceptable by the FDA in 2012, but it has yet to be adopted by the US Pharmacopoeia, a key medical standards group7. This means that any company wishing to utilize rFC testing in place of LAL throughout their drug development processes must jump through more hoops to prove the validity of their approach. To many companies, the extra costs associated with these steps are a strong deterrent; however, others are willing to pay the price to ease the environmental toll of the horseshoe crab blood harvests. For instance, biomedical research company Eli Lilly has been utilizing rFC since 2015, and in June 2020 they announced that all safety testing for human trials of one of their COVID-19 antibodies would be exclusively done with rFC12.
Unfortunately, Eli Lilly is only one company, and few others have committed to using rFC over the LAL test, even though in the nearly two decades since its original synthesis and entrance into the commercial market, numerous studies have shown rFC to be an effective, sensitive, and safe option for endotoxin testing. Environmental advocacy groups, as well as organizations promoting sustainability in technological industries have taken note of this, and over the years have continuously raised their voices to support the transition from LAL to rFC. One such voice is Ryan Phelan, who runs the nonprofit group Revive and Restore, which “supports technological solutions to conservation problems.” “It is crazy… that we are going to rely on a wild animal extract during a global pandemic,” remarked Phelan recently to the New York Times, referring to the horseshoe crab’s critical role in the global COVID-19 vaccine development efforts2,13,14. Championing a similar sentiment, last month the National Wildlife Federation called for more protections of horseshoe crab populations, with their announcement specifically urging for widespread transition to rFC in place of the horseshoe-crab-dependent LAL tests in biomedical work. “With the horseshoe crab now under threat of extinction,” said NWF executive director Curtis Fisher, quoted in the announcement6, “The development of a safe and sustainable synthetic chemical provides an alternative that allows for medical innovation while protecting this iconic species.”
Because the safety and efficacy of rFC has been demonstrated, and because the existing LAL test for endotoxins depends heavily on harvesting blood en masse from an animal whose presence is crucial to coastal ecosystems, a global switch to rFC in lieu of the LAL test is a moral imperative. Creative and sustainable innovation is the only way for biomedical research and healthcare to adapt to unprecedented challenges, and organizations forging this path rather than shirking from it will cement themselves as leaders in the field. Understandably, big shifts like this take time to build momentum, and the momentum has to start somewhere. To this end: I am calling on any readers who made it this far (congrats, almost there!), especially if you work in a healthcare or laboratory setting, to think critically about the materials you use on a daily basis. Are the materials sustainably sourced? Are there alternatives to your work materials that are more environmentally friendly? If not, is there any way for you to reuse or recycle what you use? To my fellow researchers interested in sustainability, I highly recommend looking into My Green Lab, a nonprofit aimed towards making everyday lab science more sustainable by recommending specific ways to cut down on energy usage and supply your research with greener materials. The more that bench researchers build sustainability efforts into their regular workflows, the more these practices will become ingrained in the way things are done – this is how we can begin forging a legacy of sustainability from the ground up. Change starts small, and I believe it could start here.
1. Levin J, Bang FB. Clottable protein in Limulus; its localization and kinetics of its coagulation by endotoxin. Thromb Diath Haemorrh. 1968;19(1):186-197. doi:10.1055/s-0038-1651195
2. Gorman J. Tests for Coronavirus Vaccine Need This Ingredient: Horseshoe Crabs – The New York Times.https://www.nytimes.com/2020/06/03/science/coronavirus-vaccine-horseshoe-crabs.html?algo=identity&fellback=false&imp_id=920210939&action=click&module=Science Technology&pgtype=Homepage. Published 2020.
3. Maloney T, Phelan R, Simmons N. Saving the horseshoe crab: A synthetic alternative to horseshoe crab blood for endotoxin detection. PLoS Biol. 2018;16(10):1-10. doi:10.1371/journal.pbio.2006607
4. Madrigal AC. The Blood Harvest.https://www.theatlantic.com/technology/archive/2014/02/the-blood-harvest/284078/. Published 2014.
5. Sarah Zhang. The Last Days of the Blue-Blood Harvest.https://www.theatlantic.com/science/archive/2018/05/blood-in-the-water/559229/. Published 2021.
6. Desantis M. National Wildlife Federation Calls for Increased Horseshoe Crab Protections.https://www.nwf.org/Home/Latest-News/Press-Releases/2021/6-22-21-Horseshoe-Crab-Protections. Published 2021.
7. Fox A. The Race for a Coronavirus Vaccine Runs on Horseshoe Crab Blood Pharmaceutical companies use the creature ’ s blue blood to test for contaminants.https://www.smithsonianmag.com/smart-news/race-coronavirus-vaccine-runs-horseshoe-crab-blood-180975048/. Published 2020.
9. Reich J, Heed K, Grallert H. Detection of naturally occurring bacterial endotoxins in water samples. Eur Pharm Rev. 2014;19(6):67-68.
10. McKenzie J, Alwis K, Sordillo J, Kalluri K, Milton D. Evaluation of lot-to-lot repeatability and effect of assay media choice in the recombinant Factor C assay. J Environ Monit. 2011;13(6):1739-1745. doi:10.1039/b000000x/McKenzie
11. Abate W, Sattar AA, Liu J, Conway ME, Jackson SK. 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;66(7):888-897. doi:10.1099/jmm.0.000510
12. Miller J. Wildlife groups pressure Big Pharma to curb crab blood addiction.https://www.reuters.com/article/us-health-coronavirus-pharmaceuticals-cr/wildlife-groups-pressure-big-pharma-to-curb-crab-blood-addiction-idUSKBN2382SG. Published 2020.
13. Arnold C. Horseshoe crab blood is key to making a COVID-19 vaccine—but the ecosystem may suffer. National Geographic. https://www.nationalgeographic.com/animals/article/covid-vaccine-needs-horseshoe-crab-blood. Published 2020.
14. Prudente T. How the coronavirus vaccine relies on Maryland ’ s strangest fishery : horseshoe crabs.https://www.baltimoresun.com/coronavirus/bs-md-coronavirus-horseshoe-crab-blood-vaccine-20201231-gijaccmxf5dabh4l36nzve6jre-story.html.