In drug development, all small molecules, biologics, medical devices and raw materials must be tested and proven free of endotoxin–the toxic outer layer of Gram negative bacteria, which causes inflammatory responses and illness in humans. Doing so requires “endotoxin testing.”
Enter Limulus polyphemus–the American Horseshoe Crab.
This ancient creature, which predates Homo sapiens by almost 450 million years, has an elegant, yet primitive, “immune” system which does not develop antibodies in response to infection. Instead, cells in their blue blood (yes, blue), called amebocytes, bind and inactivate bacteria by seeking out endotoxin.
An Assay is Born
In 1971, innovator and founder of Endosafe, James F. Cooper, PhD, extracted amebocytes from the blood of Horseshoe Crabs to produce an aqueous extract called Limulus Amebocyte Lysate (LAL). He then used this extract to test for bacterial endotoxins in a sample of radiopharmaceuticals.
His test turned out to be 10 times more sensitive than the current test for endotoxin. So sensitive was the test, it could detect one picogram/mL of bacterial endotoxin. One picogram (or 0.000000000001 grams) is equivalent to 1 second in 320 centuries, or one grain of sugar in an Olympic-sized swimming pool!
The LAL (Limulus Amebocyte Lysate) endotoxin assay was born.
Regulations to Protect the Horseshoe Crab
In 1972, James Cooper worked with scientists and members of the FDA to test and validate the LAL test. In 1973, the FDA announced that LAL was a biological product and subject to regulations regarding its manufacture. That same year, Cooper and others drafted a return-to-sea policy prohibiting the sacrifice of crabs after collection. And in 1980, federal regulations officially proclaimed that “horseshoes crabs must be handled in a manner so as to minimize injury, and be returned alive to their natural environment after the collection of blood.”
James and Frances Cooper Draft Legislation
Prior to 1991, there were no laws or regulations in South Carolina–our main collection site–dealing with Horseshoe Crabs. So, that year, James and Frances Cooper sat down and wrote state legislation, which provided for the management and regulation of the Horseshoe Crab fishery. The regulations were approved, stating that crabs could only be collected for the production of LAL reagents and collection could only be done by hand–greatly minimizing the harm to the animal.
In addition, many other things became subject to oversight, including how many crabs could be harvested, where they were collected, how often, how they were transported, utilized, and ultimately returned. Permit holders were subject to inspection too, and had to provide monthly harvest reports covering numbers, weights and harvest area.
Protecting Horseshoe Crabs the Charles River Way
Every year, we provide pre-harvest season training for our employees. This involves documented cGMP training, education on the ecological and biomedical significance of the Horseshoe Crab, training on all SOPs detailing the handling and preparation of Horseshoe Crabs, as well as all SOPs detailing bleeding practices.
Additionally, we have an annual meeting before the season starts. In attendance are Endosafe production managers, watermen, and representatives from the South Carolina Department of Natural Resources–who are involved in permitting, statistics, and enforcement.
We also perform annual performance reviews on our watermen, which include a review of permit requirements/conditions, a discussion of the previous year’s issues, as well as a review of plans to improve performance measures. We also assess the efficacy of our public awareness campaigns, which include posters at boat landing sites and pamphlets for public distribution.
Education efforts are constant as well. In fact, all of our operations are open to students from schools in Charleston. Classroom visits involve a conference room presentation on the ecological, biomedical, and scientific significance of the Horseshoe Crab as well as a direct observation of the Horseshoe Crab operation.
And finally, as the Endosafe scientists have refined their technology over the years, the amount of raw material needed to perform an LAL test has steadily declined. For example, the kinetic chromogenic assay (KCA) uses 50% of the raw material used in a conventional gel-clot test. The new Endosafe®-PTS uses less than 5% of the raw material of a gel-clot test.
The Population Numbers are In
So, what does this all mean for the Horseshoe Crab population? In 2009, the Atlantic States Marine Fisheries Commission (ASMFC) Stock Assessment showed that the Horseshoe Crab population indices for the Southeastern Atlantic Coast were stable or increasing. Additionally, the Sea-Map Coastal Trawl Survey, conducted by South Carolina DNR, shows a population increase over the last several years. And finally, results from a South Carolina tagging study demonstrated that bled Horseshoe Crabs are able to return to spawning beaches in subsequent years.