Is it too late to turn the tide for horseshoe crabs?
Because there is no Plan-et B…
This article discusses biotech’s ability to solve issues pertaining to the uses of endangered animals in production of therapies. This article shines a light on non-animal derived reagents being used in biotech to test for gram-negative endotoxins in vaccines.
Biotech does many amazing things for human health, but does that mean it should be at the cost of our environment and the health of our planet? The horseshoe crab has been part of Earth’s biodiversity before there was even anyone to give Earth its name. But it might not be here much longer if biotech continues to have such a high dependency on the arthropods’ blood.
We talked to Professor Jeak Ling Ding, or Lynne as she prefers to be known, about these amazing creatures on our Discovery Matters podcast episode, ‘ Old biotech and the sea’. Lynne detailed that recombinant Factor C (rFC), a reagent for the detection of bacterial endotoxins in pharmaceutical products, has changed the game in testing for gram-negative bacteria. This differs from other reagents that seek out contamination as it is non-animal derived, which has helped not only the horseshoe crab, but the food-chain including the red knots. There has been a lot of innovation to get to this point. Compared to the Rabbit Pyrogen test and limulus amebocyte lysate (LAL) test, rFC is a far better option for testing against contamination. Endotoxin test using rFC is robust, reproducible, specific and sustainable.
This development is very significant. The horseshoe crab pre-dates the dinosaurs. Dinosaurs are 65 million years old, while the horseshoe crab is around 450 million years old. What Lynne has shown is that if sustainability does not come to the forefront of the life science corporations globally and soon, then creatures, such as an arthropod, that has existed on Earth for 450 million years, will become extinct.
Before we talk about Lynne’s incredible discovery, let’s go back and talk about why the horseshoe crab is so important in biotechnology.
Despite its unassuming appearance, the horseshoe crab, a marine arthropod with a hard horseshoe-like exoskeleton (or carapace), is one of the most intriguing and special animals on the planet. These endearing creatures have had a hugely influential role in the biotech industry. They are valued for their blue blood.
The blood of these arthropods is special because, unlike human blood, oxygen is carried around their bodies through hemocyanin, which is rich in copper, making their blood blue. Amebocytes, which contain Factor C that is sensitive to bacterial endotoxin, constitute the valuable component of the blood, defending the horseshoe crab against pathogens.
The horseshoe crabs’ natural defense against pathogens makes their blood a key ingredient in the limulus amebocyte lysate (LAL) test, which ensures that vaccines, including those for COVID-19, are not contaminated with the ubiquitous bacterial endotoxins.
What does that look like, you ask?
Every healthy horseshoe crab is strapped to a metal table for bleeding and folded in half at its hinged carapace, where a stainless-steel needle pierces the pericardium (the membrane around the heart). 30 per cent of each individual crab’s blood is taken for the LAL test.
If any gram-negative bacterial (e.g., E. coli) infection, or endotoxin, is present then the limulus amebocyte lysate will clot, thus signalling that the medicine or medical device is contaminated. This process is essential when considering patient safety as instruments must be sterile and vaccines are uncontaminated by harmful bacteria.
However, the high demand for LAL for quality assurance of biomedical products means that the demand for horseshoe crab blood have risen to become unsustainable. 500,000 horseshoe crabs are harvested every year for LAL production, and a substantial number die in this process.
So, driven by her childhood fascination and love for these small creatures, Lynne spent the past 20 years trying to find a viable solution to replace the LAL test. Her endeavor led to the creation of a very simple, rapid, and standardized non-animal derived recombinant Factor C (rFC) to detect endotoxins.
This breakthrough was triggered through serendipity, one of our favorite topics on Discovery Matters.
As Lynne explained: ‘ ‘We became interested, almost like an obsession, because the LAL test kit was only produced in the United States. And it costs more than $1,000 to buy one kit which measures about 100 samples. In the mid- to late-80s, there was a group of clinician scientists who were developing the test tube baby programme in Singapore. However, the human embryos were becoming contaminated and dying prematurely. So, the clinicians suspected there was some bacteria, and they asked my colleague, Professor Bow Ho, to measure/detect the presence of gram-negative bacteria…. that was the starting point. He could only afford to purchase one LAL kit as we had minimal research grant then. The 1980’s was relatively early days for research in Singapore.’’
So, they needed a cheaper, more efficient, test for bacterial endotoxin contamination. Out of this struggle, Professor Ding’s team cloned the gene coding for Factor C, and recombinant Factor C (rFC) was born.
rFC is promoted and used by various groups. Firstly, conservationists want to ensure that the horseshoe crab will be protected and therefore promote the use rFC. Secondly, the biomedical industry uses it. Lynne explained that Eli Lilly and Pfizer directly compared LAL and rFC and found that the rFC was more efficacious and specific in detecting endotoxins and does not suffer from false positives. rFC has provided quality assurance for many advanced medicines and cures for many diseases without endangering other species.
Even as the life sciences centers around innovation, there is hope for greater sustainability. An annual survey conducted by the Center for Environmental Research and Coast Oceans Monitoring at Molloy College in 2020, recorded the highest number of horseshoe crabs in the past four years.. So, with the creation of rFC, the horseshoe crab population is recovering slowly but surely, meaning that life science organizations have an undeniably profound effect on the environment. This change is indicative of the significant role of biopharma and biotech.
 Brendan Tindall, Dogan Demircioglu, and Thomas Uhlig. “Recombinant bacterial endotoxin testing: a proven solution” BioTechniques 70 (May 2021).
 Smith DR, Newhard JJ, McGowan CP and Butler CA (2020) ‘The Long-Term Effect of Bleeding for Limulus Amebocyte Lysate on Annual Survival and Recapture of Tagged Horseshoe Crabs.’ Front. Mar. Sci. 7:607668.
 Maloney T, Phelan R, Simmons N (2018) Saving the horseshoe crab: A synthetic alternative to horseshoe crab blood for endotoxin detection. PLOS Biology 16(10).
Originally published at https://medium.com on January 27, 2022.