by Max Dakara
The ongoing pandemic has led to vaccines becoming a prominent topic of discussion for the last year or so, with the rush towards the vaccine in 2020 being one of many examples in which pharmaceuticals have relied on a particular living fossil, the horseshoe crab. These arthropods have lived for around 450 million years and as a result of evolution, it has developed an impressive immune system.
So what exactly does the blue blood of these crabs provide us with? Well, it’s the reason that pharmaceuticals entering into a patient’s body are sterile - from IVs to vaccines, from medical devices that require implantation to medicinal drugs, or any other pharmaceutical - it is important for it to be free from bacterial contaminants. The horseshoe crab contains Limulus amebocyte lysate (LAL) which is the basis of the LAL test used to check if a substance is sterile or not.
Endotoxins are present on the surface of certain bacterias such as E. coli and are pyrogens, fever-causing agents. Trace amounts of endotoxins in pharmaceuticals such as vaccines can lead to septic shock and in worst cases death, which makes LAL integral for a lot of medical procedures. The LAL test is highly sensitive to these endotoxins and allows medical personnel to be certain about whether or not the pharmaceutical is suitable for use.
The LAL test works through a series of biochemical reactions. When exposed to endotoxins, the Factor C present in LAL induces the activation of Factor B causing the activation of a clotting enzyme. These enzymes coagulate the blood, forming a gel-like substance indicating a positive result - a presence of an endotoxin.
Despite the revolutionary discovery of the blue blood’s application by Frederik Bang in the 70s, it poses a major threat to the environment. Horseshoe’s are drained of their blood and returned to the waters, this process leaves the crabs close with significant injuries and leaves them with a greater likelihood of death. The overall effect of this industry has left horseshoe crab populations to deplete and as a result disrupted the food chain of local ecosystems. Populations of animals such as the endangered diamondback terrapin who rely on their eggs for sustenance are among the few that are affected as the industry grows larger.
The high price of the blue blood at $15,000 per quart (0.95L) as well as the damage to ecosystems on the North American coastline led to a synthetic alternative to be thought of and developed. This idea for an alternative was thought of by Ling Ding Jeak and Bo How, scientists working at the National University of Singapore, who successfully created one in 2003. Known as recombinant Factor C (rFC), it uses a protein responsible for the production of Factor C, the highly sensitive component of the LAL that detects endotoxins. Detection of an endotoxin activates the rFC as a result, it cleaves a fluorogenic substance causing the solution to become fluorescent. A machine then measures the level of fluorescence from the substance.
This alternative however hasn’t been met with widespread acceptance especially by the US’ FDA and only recently was it approved by the EU and added to their pharmacopeia in 2016. This is due to the fear of having a test that isn’t as sensitive as LAL, which could lead to patient death if a substance is shown to be sterile when it’s contaminated.
However, several studies have been done to evaluate its efficacy and have shown that it has a high sensitivity similar to LAL, obtaining results that are consistent and reliable. It’s more specific towards endotoxins and bacteria unlike LAL, which is susceptible to false positives from a detection of β-glucans. rFC being synthetic allows it to be more uniform throughout different batches, unlike LAL where horseshoe crabs of different ages, genders, environment, and other factors would cause a variation from batch to batch. Other advantages are that it’s likely to be much more affordable to produce than LAL and conversion to rFC would lead to a decrease in the death of horseshoe crab species in both North America and Asia due to the forecasted 90% drop in demand for their blue blood.
There have been pharmaceutical companies such as Lonza and Hyglos that have taken up the technology and produced a test. This is because they view it as a more sustainable practice and as a way to modernize medical practices and tests. As more and more pharmaceutical companies and regulatory bodies acknowledge rFC to be either on par or much better by seeing the results, then a change towards a more sustainable source of bacterial testing can come about.
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