THE HORSESHOE CRAB

(BBC from wikipedia)

Vaccines Rely on Horseshoe Crab’s Blue Blood for Purity

Every spring, 500,000 Atlantic Horseshoe Crabs are collected by four biopharmaceutical companies as they crawl to the mid-Atlantic beaches to lay their eggs.  The helmet-shaped arthropods are placed upside down on a rack, and a sterile needle is inserted near the heart to collect about 30 percent of their blood.1 2  The crabs are returned to the ocean and their blood, often referred to as “blue gold” due to it’s color and worth of $60,000 per gallon in a global industry valued at $50 million per year, is used to purify vaccines, injectable drugs and other sterile pharmaceuticals.3 4

Unique Horseshoe Crab Blood Reacts to Endotoxins

Commonly referred to as a “living fossil,” horseshoe crabs have an open circulatory system with no adaptive immune response.  Instead, they have survived unchanged for an estimated 200 million years through an innate immunity based on granular amebocytes in their blood.5 These amebocytes contain the proteins of its blood clotting system, which are released when they come in contact with unwanted organisms like Gram negative bacteria.6 Endotoxin is a lipopolysaccharide found on the outer membrane of Gram-negative bacteria (such as E. coli, Salmonella, Shigella, Neisseria, Haemophilus influenza, and Bordatella pertussis)7, and when it contaminates vaccines and other injectable pharmaceutical products it can cause fever, shock, organ failure and death.8 The coagulation proteins extracted from horseshoe crab blood, called lysates, produce an instantaneous, visible reaction to endotoxins, which has driven commercial demand from pharmaceutical and biomedical companies to confirm drug and medical device safety.9

Even an iota of endotoxin can prove to be fatal.10 John Dubczak, General Manager at Charles River Laboratories, which manufactures and globally distributes lysate products, reports, “Detection is down to one part per trillion, but we can take it down to a tenth of a trillion, and further orders of magnitude more sensitive.”11

Synthetic Alternative Not Available

From the 1940s to the 1970s, rabbits were used to screen pharmaceutical products for endotoxins.  In 1956 medical researchers Fred Bang and Jack Levin observed that horseshoe crab blood clotted and formed a solid mass in the presence of endotoxin, leading to the development of an endotoxin assay known as the Limulus amebocyte lysate (LAL) assay, which was approved for use by the FDA in 1977.12 13 In Asia, there is a similar test called TAL (Tachypleus amebocyte lysate) which takes its name from an Asian species of crab, Tachypleus tridentatus.

In response to concerns about dwindling populations of horseshoe crabs, the time required to catch and harvest blood from crabs, and variability that occurs with pooled batches of harvested animals, new endotoxin tests using synthetically produced recombinant Factor C (rFC) were developed to relieve pressures of a growing demand for the LAL assay.14 Although this synthetic rFC alternative was approved for use in Europe in 2016, the American Pharmacopeia, which sets the scientific standards for drugs and other products in the U.S., on June 1, 2020 declined to place rFC on equal footing with crab lysate, claiming that its safety is still unproven.15

Horseshoe Crabs An Endangered Species

Horseshoe Crabs can take ten to twelve years to reach maturity, and only three out of 100,000 survive their first year of life.  Once they reach adulthood, the crabs have few non-human predators and are expected to live ten or more additional years.  It is only once they reach maturity that they begin their spring migration from the shelter of deeper waters to their spawning beaches.  In the U.S., mortality of horseshoe crabs due to LAL production is estimated to be relatively low—8 to 15 percent.  However, in Asia, mortality is 100 percent because after bleeding, the crabs are sold to secondary markets for food and chitin production.16

As a result of overharvesting for use as food, bait and biomedical testing, and because of habitat loss, the American horseshoe crab is listed as Vulnerable to extinction and the Asian tri-spine horseshoe crab is classified as Endangered on the International Union for the Conservation of Nature (IUCN) Red List of Threatened Species.  The two additional Asian horseshoe crab species will soon be listed on the IUCN Red List.17

In 1990 biologists estimated 1.24 million American horseshoe crabs spawned in Delaware Bay, a main egg-laying spot and prime collection point for companies.  By 2002 that number had dropped to 333,500, and have remained at that level.18  Throughout the 1980s and 1990s biomedical harvesting seemed sustainable, and the pharmaceutical industry claimed that only three percent of the crabs they bled died.  However, by the 2000s, annual horseshoe crab counts during spawning season revealed smaller numbers and a 2010 study found that as many as 30 percent of the bled crabs ultimately died—ten times as many as first estimated.19

The depletion of horseshoe crabs can become a weak link in the food chain that reverberates with potentially disastrous consequences for species that rely on horseshoe crab eggs as vital food sources, such as diamondback terrapins, striped bass and flounder, and many migratory birds.

Global Demand for Vaccines and Medical Devices Rising

Demand for lysate assays to test biomedical devices and vaccines is likely to rise significantly in the next two decades due to population growth and aging, and medical advancements to improve or prolong life.  Any product or device that comes in contact with blood, lymph, spinal fluids or mucus membranes that do not pass through the body by an oral pathway must be tested for the presence of endotoxins.

Although vaccines are a small segment of the global pharmaceutical market, they represent the fastest growing segment of this industry and are heavily reliant upon endotoxin detection.20 This growth of consumer demand places pressure on the diminishing horseshoe crab resource needed for endotoxin detection.

“All pharmaceutical companies around the world rely on these crabs.  When you think about it, your mind is boggled by the reliance that we have on this primitive creature,” says Barbara Brummer, state director for The Nature Conservancy in New Jersey.21

Article and References: NVIC

(Here’s how we’ll know when a COVID-19 vaccine is ready.)

NATIONAL GEOGRAPHIC ARTICLE

Horseshoe crab blood is key to making a COVID-19 vaccine—but the ecosystem may suffer. Conservationists worry the animals, which are vital food sources for many species along the U.S. East Coast, will decline in number. nat'lgeographic

Each spring, guided by the full moon, hundreds of thousands of horseshoe crabs clamber onto beaches across the U.S. mid-Atlantic to lay their eggs. For hungry birds, it’s a cornucopia. For drug companies, it’s a crucial resource for making human medicines safe.

That’s because these animals’ milky-blue blood provides the only known natural source of limulus amebocyte lysate, a substance that detects a contaminant called endotoxin. If even tiny amounts of endotoxin—a type of bacterial toxin—make their way into vaccines, injectable drugs, or other sterile pharmaceuticals such as artificial knees and hips, the results can be deadly.

“All pharmaceutical companies around the world rely on these crabs. When you think about it, your mind is boggled by the reliance that we have on this primitive creature,” says Barbara Brummer, state director for The Nature Conservancy in New Jersey.

Every year, pharmaceutical companies round up half a million Atlantic horseshoe crabs, bleed them, and return them to the ocean— after which many will die. This practice, combined with overharvesting of the crabs for fishing bait, has caused a decline in the species in the region in the past few decades.

In 1990, biologists estimated 1.24 million crabs spawned in Delaware Bay, a main egg-laying spot and prime collection point for the companies. By 2002, that number had dropped to 333,500. In recent years, numbers of Delaware Bay spawning crabs have hovered around the same amount, with the 2019 survey estimating about 335,211. (The pandemic canceled the 2020 crab count.)

Catching crabs and harvesting their blood is time-consuming, and the resulting lysate costs $60,000 per gallon. In 2016, a synthetic alternative to crab lysate, recombinant factor C (rFC), was approved as an alternative in Europe, and a handful of U.S. drug companies also began using it.

But on June 1, 2020, the American Pharmacopeia, which sets the scientific standards for drugs and other products in the U.S., declined to place rFC on equal footing with crab lysate, claiming that its safety is still unproven.

Starting in July, Swiss-based Lonza will begin manufacturing a COVID-19 vaccine for human clinical trials—and they’ll have to use lysate in the vaccine if they plan to sell it in the U.S. (Here’s how we’ll know when a COVID-19 vaccine is ready.)

Human health and safety, especially for something as high stakes as the coronavirus vaccine, is paramount, says Brummer. But she and other conservationists fear that without rFC or other alternatives available, the ongoing burden on horseshoe crab blood for COVID-19 vaccines and related therapeutics may imperil the crabs and the marine ecosystems that depend on them.

A written statement from Lonza says that testing the company’s COVID-19 vaccine will not require more than a day’s worth of lysate production from the three U.S. manufacturers.

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Lysate: a preparation containing the products of lysis of cells [a substance that detects a contaminant called endotoxin]

Lysis: Lysis is the breaking down of the membrane of a cell, often by viral, enzymic, or osmotic mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a lysate. Wikipedia

Endotoxin: What are the effects of endotoxins?

Control of cell product contamination with endotoxins during the manufacturing is important for two reasons. First, endotoxins are dangerous when entering the blood, causing fever and a wide range of other possible effects including aseptic shock and death.