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Will a Shorebird Knot Up Bacterial Endotoxin Assay Supplies?

Aug 22, 2014

The remarkable red knot shorebird has one of the longest migrations of any bird—over 9,000 miles from the southern tip of South America to the Arctic. That’s 18,000 miles round trip. A long annual journey made possible by its many stops on South American and North American Atlantic shores.

The last stop on its northern return to its Arctic breeding grounds along the coasts of Delaware, New Jersey and New York is perhaps the most pivotal, for there these birds fill up on enough fuel to power them north to their summer mating grounds. The food, eggs. Horseshoe crab eggs. And thus, the migratory pattern of this little shorebird is inextricably linked to the lives of fish farmers, fishermen and users of intravenous pharmaceutical products and medical devices worldwide.

Countless numbers of patients rely on intravenous pharmaceutical and medical device products to manage illnesses, cure and prevent disease and to maintain their health. These products must be tested to assure no harmful bacteria are present. The ubiquitous test is the Limulus amebocyte lysate (LAL) method. Of course, LAL is extracted from another remarkable creature, and one of the most distinctive ones found in the Atlantic Ocean, Limulus polyphemus—the horseshoe crab.

Most patients have no idea when they receive an injection or a medical device that a horseshoe crab (Limulus) had to be bled in order for a testing lab to conduct the LAL pyrogen test. The crab’s blood cells (amebocytes) contain the active components of the reagent. The process for producing the reagent utilizes lysed blood cells (lysate).

Are the health needs of countless patients at loggerheads with the existence of this little bird?

LAL, A Disruptive Technology

Prior to the 1970s, labs tested for pyrogens in products by injecting rabbits. Switching over to LAL was not only the humane thing to do, it was easier, faster and cheaper.

LAL testing was pioneered at the Marine Biological Laboratory in Woods Hole, Mass. by Dr. Jack Levin, a hematologist at The Johns Hopkins University, who had a particular interest in human blood platelets. Professor Frederik Bang, MD, also of Hopkins, was at the Marine Biological Laboratory studying horseshoe crabs and called on the the hematology department to send an expert to help explain why the crabs’ blood clotted.

“Joining Dr. Bang was one of the smartest professional decisions I ever made in my life,” Dr. Levin told the PDA Letter.

“In studying the coagulation [of the blood of the horseshoe crab], I quickly discovered that I could not stop the blood from clotting using the usual anticoagulants,” he continued. “This caused a great limitation in what I could initially do. One day, in desperation, I considered that endotoxin was triggering clotting. I prepared glassware which was endotoxin free, and discovered I could stop coagulation from occurring, and that was an enormously important clue that in fact bacterial endotoxin was capable of clotting Limulus blood.”

Thus, a truly superior test was created. By switching over to it, healthcare companies of all stripes gained a new public image, as rabbit and other kinds of animal testing were growing unpopular.

LAL testing involved an animal of little public interest; it did not require the killing of horseshoe crabs nor the breeding of them in controlled environments. Crabs chosen for bleeding were removed from the water, bled and then returned to their habitat. When handled correctly and promptly returned to their habitat, horseshoe crabs never die for LAL purposes. Dr. Levin said that in all his years of bleeding crabs, his work never resulted in a death.

The development and spread of LAL testing is a remarkable and almost unprecedented example of a new technology creating sweeping changes in both the public view and the bottom line of multiple industries in such a short period of time.

Alternative Tests Emerge

The success of this test, however, relies on an easily accessible supply of horseshoe crabs. Not a problem in the 1970s, but by the 1990s, increasing demand for these ancient animals from healthcare and commercial fishing, along with environmental damage and warming seas, meant the horseshoe crab was no longer as readily available.

In fact, Limulus polyphemus is the only one of four species of horseshoe crab—the only species in North America—not currently threatened. Populations of the three Asian species—Carcinoscorpius rotundicauda, Tachypleus gigas and Tachypleus tridentatus—have declined rapidly. Limulus polyphemus, however, is under threat, too. And there are no horseshoe crabs elsewhere. Not on European, Australian or African coasts. The type of amebocyte lysate (AL) used depends on the source blood, so besides LAL, there is CAL and TAL.

Understanding the threat to these creatures, particularly those in Asia, National University of Singapore Professor Jeak Ling Ding, with an eye to the dangerously dwindling populations of the Asian species, decided to explore the possibility of developing a completely environmentally friendly alternative to pyrogen testing.

Dr. Ding also believed that a more consistent test could be created rather than the one that relies on LAL. She noted in a conversation with the PDA Letter that there are “batch-to-batch variations in sensitivity and specificity of pyrogen detection.” These variations occur, she explained, as a result of habitat and seasonal changes.

“Furthermore, LAL/TAL contains Factor G, which is sensitive to fungal toxins, resulting in the activation of the coagulation cascade,” said Dr. Ding. This can result in “a false positive test result for pyrogen.”

Following many years of “fundamental research on the basic biochemistry and molecular biology of endotoxin-activated coagulation cascade reactions in horseshoe crab amoebocyte lysate by many researchers including our own,” Dr. Ding and her team had “a good understanding on how Factor C—the first enzyme in the cascade—is the target of endotoxin.”

Based on the research, CAL was chosen for genetic engineering because it is the most potent endotoxin-sensitive lysate, as a result of its microbe-rich estuarine habitat.

After nearly two decades, however, recombinant Factor C (rFC) and other LAL alternatives have not caught on.

Lonza, which markets PyroGeneTM, an rFC pyrogen test has seen sales increase recently, but nowhere near enough to suggest LAL is out of favor.

Lonza Walkersville’s Allen Burgenson, who is the President of the PDA Capital Area Chapter, recently discussed LAL testing, the stresses on horseshoe crab populations and the alternative test PyroGeneTM with the PDA Letter.

One reason for the slow uptake of PyroGeneTM, he said, is because it is not a compendial test. “It’s not yet found in the United States Pharmacopoeia, the European Pharmacopoeia, or the Japanese Pharmacopoeia,” said Burgenson.

Alternative tests are acceptable, however, and the U.S. FDA went so far as to specifically name rFC and the monocyte activation type pyrogen test as acceptable alternatives to USP <85> BET Testing in a 2012 Q&A guidance (1), as long as their use is validated per USP <1225>.

Burgenson noted that once a firm validated the test for a few of its products, it can “write a comparability protocol and submit it to the FDA.” Application of the test to additional products “instead of being a prior approval submission the way the first couple would have been, they become a CBE-30, with changes being effective in 30 days once the protocol is approved by FDA.”

While other suppliers of LAL like Charles River Laboratories International (2) do not agree with environmental/preservation arguments for moving away from LAL, Lonza—which incidentally holds an exclusive license with the developers of rFC—believes there are ethical reasons to switch to alternative tests.

Burgenson explained: “The horseshoe crab is obviously a live animal, and in Europe, there is a mandate to remove animals from the manufacturing and testing process wherever possible.”

Threatened LAL Supplies

In addition, he cited potential threats to Atlantic horseshoe crab populations beyond overfishing by the fishing and food industries, the damage from which seems to have reversed in recent years.

“Right now, we seem to have reached a point of stability. There was a point several years ago where the population was severely impacted by overfishing, and the Atlantic States Marine Fisheries Commission had to restrict that and to increase the population. And it looks like the population is recovering,” Burgenson said. However, “as you saw with Superstorm Sandy when it came ashore in New Jersey and Delaware, the beaches where the horseshoe crabs spawn were really torn up, so Mother Nature can have a severe impact, especially with storms such as that on the breeding grounds—spawning grounds—so the horseshoe crabs can’t get to shore to spawn, [and] that can severely impact the population of horseshoe crabs in that area.”

And demand for LAL could outpace the supply, Burgenson predicts. “Well, as the pharmaceutical industry grows, it increases the greater demand on the supply of LAL....For every two products that make it to market, 98 don’t. But you still have to test all of those products as they are being developed and put through the submission pipeline. So, there’s a lot of products that never make it to market that still need to be tested. So, as that demand increases, supply of LAL or the population of horseshoe crabs is stationary. The population is not increasing in great numbers.”

Finally, the greatest possible threat to LAL supplies is not overharvesting or ecological disaster. Rather, it is the red knot shorebird.

Burgenson has followed the plight of the shorebird closely, and even appeared on a recent American Public Media Marketplace segment to discuss it (3).

“Right now the red knot is threatened, and it is being proposed to be put on the Endangered Species List. This bird flies all the way from Tierra del Fuego at the very tip of South America all the way to the Arctic every year to breed, and there is a stopover in the Delaware Bay region where they gorge themselves on horseshoe crab eggs. Horseshoe crabs spawning and the arrival of these birds happens simultaneously—while, it actually happens up and down the East Coast—and once the birds get heavy enough to continue their journey to the Arctic, they’ll fly and go up to the Arctic for breeding.”

If listed as a “threatened species” under the U.S. Endangered Species Act, “the whole objective is to try to reduce any stress on the horseshoe crab population to keep those birds healthy and existing,” said Burgenson.

Dr. Levin, who does not consider the alternative methods to be as useful or effective as LAL, believes the horseshoe crab conservationists finally have a powerful ally in their efforts to stop harvesting for bait—the National Audubon Society. It remains to be seen, however, what impact such listing would have on harvesting for LAL purposes, since the crabs are left relatively unharmed.

The public comment period on the proposal to list the red knot as an endangered species was reopened in April 2014 and closed in June. A decision could be made by September. It is unclear as of now how the decision will impact horseshoe crab harvesting, if at all.

While the red knot’s potential status as a threatened species might not impact the ability of LAL suppliers to bleed horseshoe crabs, it does serve as a cautionary note to healthcare companies that rely on living creatures for critical materials. With environmental strains, growing demand for food (animal and human), and the desire to preserve species, companies must vigilantly monitor supplies and have backup plans in case supplies are disrupted.

In the case of LAL, alternative methods are available.

References

  1. Guidance for Industry Pyrogen and Endotoxins Testing: Questions and Answers, U.S. Food and Drug Administration: June 2012 www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM310098.pdf
  2. Charles River Laboratories. “Charles River Horseshoe Crab Conservation.” criver.com. www.criver.com/microsites/horseshoe-crab-conservation (accessed August 18, 2014).
  3. Allen Burgenson, interview by Sabri Ben-Achour, Marketplace, American Public Media, June 16, 2014. www.marketplace.org/topics/sustainability/paying-save-nature-could-mean-win-everyone

About the Experts

Allen Burgenson is the Regulatory Affairs Manager for Lonza Walkersville Inc. He holds a MS in Biotechnology Management from the University of Maryland, and serves on the Horseshoe Crab Advisory Panel of the Atlantic States Marine Fisheries Commission.

Dr. Jack Levin has been a member of the faculty of the University of California School of Medicine in San Francisco since 1982. Previously, he was Professor of Medicine at The Johns Hopkins Univ. School of Medicine, Baltimore, Md.

Jeak Ling Ding, PhD, is a professor of biological sciences at the National University of Singapore. Her research interests focus on innate immunity and pathogen surveillance strategies. She received her doctorate from the Royal Postgraduate Medical School in London.

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