S2, E08: The Life (Saving) Blood of Horseshoe Crabs

 

About This Episode

The discovery of limulus amebocyte lysate (LAL) in the blood of horseshoe crabs has forever changed bacterial endotoxin testing. Today, researchers continue to look for any way they can protect these seafaring creatures while refining testing methods to decrease the need for animals. Join our panel for an in-depth look at how they’re working with legislators to preserve the horseshoe crab population and how LAL testing is the safest in vitro method to detect harmful bacteria.

  • Episode Transcript

    Chris (00:06):
    Hello, and welcome to Vital Science.

    If you've ever spent time in the warm months visiting the many beaches and estuaries along the U.S. Eastern Seaboard, you might have crossed paths with a rather large and alien looking species - the horseshoe crab. Known less commonly by its scientific name, Limulus polyphemus, the Atlantic horseshoe crab is remarkable for numerous reasons. The species has stood the test of time, thriving in the ocean for the past 450,000,000 years. But it has only been developments of the past several decades that have brought the crab into the spotlight.

    If you've ever spent time in the warm months visiting the many beaches and estuaries along the U.S. Eastern Seaboard, you might have crossed paths with a rather large and alien looking species - the horseshoe crab. Known less commonly by its scientific name, Limulus polyphemus, the Atlantic horseshoe crab is remarkable for numerous reasons. The species has stood the test of time, thriving in the ocean for the past 450,000,000 years. But it has only been developments of the past several decades that have brought the crab into the spotlight.

    And so today, we welcome two horseshoe crab experts who delve deeper into this remarkable creature, explain the evolution of LAL in vitro testing, describe ongoing conservation efforts, and discuss current research on LAL alternatives. With that, I'll turn it over to Gina.

    Gina (01:24):
    Hello, and welcome to another episode of Vital Science. Thanks for joining us.

    Limulus amebocyte lysate, or LAL, is used to detect endotoxins in all injectable pharmaceutical products and implantable medical devices. If endotoxins make it into the bloodstream, a patient can develop a pyrogenic response or fever, and even symptoms of septic shock. Thanks to LAL and horseshoe crabs, we all have received endotoxin-free products over the years. Why should we thank horseshoe crabs? Well, stay tuned to find out what horseshoe crabs have to do with keeping us safe from endotoxins, and how we are keeping them safe from endangerment.

    In this episode, we will be discussing endotoxin testing, horseshoe crabs, and sustainability. Our guests today are Dr. Norm Wainwright, senior director of research and development, and Nicola Reid, associate director of product development with Charles River. Both have worked on evolving LAL testing to make it more efficient for pharmaceutical companies, safer for patients, and more sustainable for the planet.

    Welcome, Norm and Nicola!

    Nicola Reid (02:34):
    Hello.

    Norm Wainwright (02:35):
    Good morning.

    Gina (02:36):
    Hey! So let's start with you, Norm. Can you share a bit about your background with us?

    Norm Wainwright (02:42):
    Yes, I grew up on the New Jersey shore. So my first contacts with horseshoe crabs actually came probably when I was six or seven years old. Our family would frequently go boating and lunchtime, we'd just pull over and have a sandwich or two while we waited around in the shallow water. So seeing a horseshoe crab at that age with bare feet was a little bit startling, but it was a very positive thing in retrospect.

    Gina (03:14):
    And did that somehow imprint upon you to make some decisions in your future career?

    Norm Wainwright (03:21):
    Well, I guess it did. Although at the time, it probably wasn't first thing on my mind. All living things are beautiful in their own way and I think horseshoe crabs are beautiful in a very unique way. They're very alien looking and I think your first encounter is a bit startling, to say the least. But it is kind of funny how, later in my career, after quite a bit of biochemistry training, molecular biology, the opportunity arose to go to the Marine Biological Lab and work with horseshoe crabs and really have some direct contact with these relatively strange looking creatures.

    Gina (04:07):
    Wonderful. And now for you, Nicola, I suspect you're not able to see horseshoe crabs on the shores as often as Norm and I are. Tell us a little bit about yourself.

    Nicola Reid (04:18):
    Absolutely, Gina. So I'm based in the United Kingdom, so we don't have the luxury of having horseshoe crabs in our water. I started to work with Charles River 18 years ago, but my first experience with horseshoe crabs was with actual endotoxin testing a few years before that when I started to work in pharmaceuticals. So I learned the understanding of where LAL come from from these amazing creatures, but I didn't get the opportunity to actually see one in the flesh, so to speak, until I started to work for Charles River. It's funny what Norm says that when you actually see them for the first time, it's quite daunting. They look like a very unusual creature, but they're just absolutely amazing the way they function, and I know we can talk a little about that today, and the way they help to protect us all from a medical perspective, also.

    Gina (05:11):
    Wonderful. Thanks for that, Nicola. And tell us a little bit about what you do in your role. What are some of your responsibilities and things you're working on day to day?

    Nicola Reid (05:21):
    Absolutely! So I work in a product management team and we work very closely with R&D and product development. So we're responsible for looking for new products and innovations and how we move LAL or endotoxin testing forward both now and in the future, and how we improve upon the products that we currently have out there in the market. So we're continuously looking to improve on the testing itself, on the software that we have, on the hardware that we have, to meet the demands and the needs of the pharmaceutical companies.

    Gina (05:52):
    Now, Norm, everyone I speak to about horseshoe crabs is always surprised by the important role they play in biomedical research. Can you tell us a little bit how that came to be?

    Norm Wainwright (06:05):
    Sure. As I mentioned, I did spend some time with the Marine Biological Lab in Woods Hole, and one aspect of that facility, a hallmark, really, is that they have explored marine invertebrates, particularly, and how they interact with human health. How they might be used as models to understand human health a little bit better. So for instance, things like how nerves get transmitted electrically was discovered by looking at a large bundle of nerves in squid and where they could take some readings.

    So for the horseshoe crab, there's really two areas that came to play here. One was vision - their eyes have a very convenient nerve that one can look at to study vision and how light gets transmitted to nerves, transmitted to the brain, etc. And the second is the clotting reaction in response to bacteria, and that's really what led to LAL. Investigators at the Marine Biological Lab, specifically Frederick Bang out of Johns Hopkins, and Jack Levin, started working with horseshoe crabs as a model to understand bacterial infections. Now remember, when they injected bacteria into a horseshoe crab, it was rather a dramatic clotting of the blood that occurred, which really puzzled them and caused them to start asking totally different questions than what they initially were setting out to do.

    So after actually several years of experimentation, they discovered that this mechanism was the bacterial endotoxin present in the cell wall of bacteria that the horseshoe crab blood cells were reacting to, basically triggering a clotting reaction as a way of their defense mechanism, how they might deal with an injury that would bring bacteria into contact with their blood, and so on. So by this, by a bit of serendipity, the clotting mechanism was discovered, its relationship to bacterial endotoxin, and as you mentioned in the beginning of this podcast, bacterial endotoxin can be very toxic when it's exposed to the blood of humans in even small concentrations.

    Gina (08:56):
    That really is a perfect way to describe it. Serendipity, I love that. What an interesting story and the science continues to amaze me every day.

    Nicola, what Norm is describing, I take it eventually led to this LAL test that I mentioned before. Can you tell us a little bit about that test and how it's evolved? What's been exciting to you in that evolution?

    Nicola Reid (09:21): Around that time when the scientists were discovering that there was actually a clotting reaction cascade, so to speak, when in the presence of endotoxin, what was being used for endotoxin testing within pharmaceuticals at that time was rabbits, which is known as the rabbit pyrogen test. So the products would be injected into the rabbits and we'd be looking for an increase in temperature over so many rabbits to declare a positive. Of course, nobody wants to be using animals, animal models, so when Dr. Bang and Dr. Levin discovered this, then this opened up a whole new scope of, "Could we use something? Could we have an in vitro method here for endotoxin detection?"

    It wasn't quite that simple. There's a lot needed to be done, of course, in order to kind of perform an assay, and many people were involved in that process ... way, way back now ... a number of decades ago to put together what was Limulus amebocyte lysate, which is LAL, what we use today in the assay forms. And obviously this comes from the blood of the horseshoe crab. So we were able, it took a long time to do comparative studies from rabbit pyrogen testing to LAL to have the confidence that this new LAL was as good as rabbits. Actually, what turned out was it was better than rabbits. It was far more sensitive than the rabbit test and far better at picking up very small amounts of endotoxin. It came directly from the horseshoe crab.

    So we evolved from utilizing rabbits into this in vivo assay with LAL. And obviously, as we'll discuss today, we've continued to evolve that. That first test that we had for LAL was gel clots. Now we have numerous different tests and ways to reduce the amount of LAL as well, so we continue, it continues to evolve. But being able to move away from rabbits into this was an amazing part of science at the time.

    Chris (11:24):
    As Nicola mentions, there were many stages and players involved in the process of proposing and validating the LAL-based bacterial endotoxin test. Over time, research proved that LAL could detect as little as one picogram of bacterial endotoxin per milliliter, which for reference is the equivalent a grain of sand in an Olympic-sized swimming pool. It's pretty amazing.

    It's no surprise, then, that the test gained regulatory acceptance and it was approved as an in vitro alternative to the rabbit pyrogen test in 1977. This, of course, was for true in-the-dish kinetic plate incubation and gel clot testing where reconstituted LAL is mixed with the pharmaceutical samples. If a clot forms, the sample is contaminated. While a great improvement over the lengthy, expensive, and labor intensive rabbit test, traditional LAL testing still had its challenges, like the need for technician training, and the possibility of related user error, and the requirement for in-lab testing. These factors have driven further developments in the BET since then.

    Let's listen to how testing has evolved.

    Gina (12:31):
    And maybe this is a question for Norm. I understand with the evolution of this test, the latest assay uses cartridges instead of the traditional approach of microplates or, kind of, in the tubes. Could you tell us a little bit about those advances using cartridge technology?

    Norm Wainwright (12:53):
    Sure. As Nicola mentions, the test became very regimented in the way that LAL was able to be transmitted into an in vitro test that could be done in test tubes, originally, and then other types of small devices. But it was always a very difficult test to do in the sense that the technician had to be very careful not to get contamination from the environment near the test. So when we were looking at developing a system basically to use a small computer, handheld, that would be able to run the test without very much human intervention, it eliminates some of those problems of contact with PETs and tubes and so on.

    We developed a way to try very small amounts of the LAL reagents into a cartridge about the size of a microscope slide, a few inches long, and have the computer run the test by putting the sample in, rehydrating the sample of the enzymes from the LAL test, as well as a chromogenic substance that would change color during the course of the reaction. So we realized that we could get by with very much smaller quantities of material and, in the end, ended up with only about 5% of the amount of reagents used for such a test.

    Nicola Reid (14:35):
    It's a 95% reduction in the amount of reagent used in comparison with what we call traditional assays, those being gel clots or traditional connected plate methods. So it brought about many advantages. There was speed, there was ease of use, but I think the most important thing here is that it allowed us to use a much smaller amount of the raw material, and that's really important when we're looking at how we actually gather this material via the bleeding of these horseshoe crabs.

    Norm Wainwright (15:09):
    The other thing, that the technology allows us to make the test more portable. That one could take this handheld reader with a cartridge to any location, say, on a factory floor or even in the outside environment to perform the test that was traditionally done in the laboratory.

    Gina (15:33):
    Okay, great! Really insightful from both of you.

    One thing you said, Nicola, that I wondered if we could dig into a little bit more is about the reduction of the amount of raw materials because I know that's one of the things that Charles River focuses on heavily, is its conservation efforts of the horseshoe crabs. And I wondered if you could tell us a little bit about those things or, Norm, if you have some perspective on that because I think that's something that's probably important to mention here.

    Norm Wainwright (16:03):
    I can add that from the very beginning of the Endosafe division, which was started by Dr. Jim Cooper, that he worked very hard to work with the legislators at the state level to get laws in place that would protect the horseshoe crab, actually prevent them from being taken for the bait industry, which would have a drastic implication for the population, and really limit the collection of horseshoe crabs in a very controlled way to only be used for the biomedical application and the production of LAL.

    Nicola Reid (16:37):
    Yes, Norm, It's very important to us that we have those practices in place. We have that protection order in place certainly in South Carolina to ensure that the horseshoe crabs can only be used for biomedical purposes and not for bait purposes. But we also have other legislation in place for the welfare of the horseshoe crabs so when they are collected, we ensure they are being looked after and that they're being taken back to where they came from within a very short space of time.

    And this is really important for all people who are collecting these for biomedical purposes, to ensure they're following these welfare practices. And there are a number of people who enforce these welfare practices but also keep an eye on the numbers and the stock levels of the horseshoe crabs, and they regularly bring out reports, stock reports, to let us know how well the population is doing. And a recent report has come out and it's wonderful to know that the stock levels of horseshoe crabs are very healthy at the moment and very robust, certainly in all the areas where we currently take them for biomedical purposes.

    Norm Wainwright (17:44):
    Yes, for sure. It's quite interesting from the standpoint of the public. And I'm always a bit surprised by this, that there's such a positive connection. There are many people that just love horseshoe crabs and, even though they are really not anything like a fuzzy puppy or panda bear, but there's a genuine connection where people really appreciate having the crabs out in the environment and just being able to see them.

    So we've always been aware that the scientific information of what we do is important for the public to understand. We've actually partnered with several aquaria around the country starting in South Carolina, but later with Mystic Aquarium and certainly Rutgers University. One aspect of the Rutgers plan that I became very interested in back in, I believe around 2017 or '18, was that they were able to take some fertilized horseshoe crab eggs, and these are the eggs that are laid by the crabs in the spring in estuaries along beaches, and take some of those fertilized eggs into an aquaculture facility. Rutgers has a very sophisticated facility in Cape May, New Jersey that they use for this purpose.

    So in 2017, I believe, was the first time that Rutgers raised several of these horseshoe crabs to an age that would be allowing them to survive better in the environment. Normally, when eggs are laid on the beach by the many millions, the number of crabs that result from those fertilized eggs, it's quite small. The eggs are eaten by sea birds, by various fish, and the amount of crabs making it to the adult stages are really quite small. So what Rutgers did by taking the fertilized eggs into aquaculture is to actually protect them, allow them to grow up a little bit more, four months or six months, and then release them back where they could better survive with the idea that this would be a way to enhance the natural populations to basically do a bit of stock enhancement for the horseshoe crabs. This is something we have begun partnering with them to help support that work and that continues this day.

    Chris (20:35):
    The horseshoe crab protections that Endosafe founder Dr. James Cooper wrote and convinced South Carolina to adopt into law have served as a foundation for preservation efforts that continue today in new regions, like the northeast. If you'd like to dive deeper into horseshoe crab conservation efforts, examine population data, or learn more about some of the programs that Charles River has been involved in, we invite you to explore the resources we've collected in the show notes.

    In our next segment, our guests cover what's perhaps on everyone's mind: How is LAL collected? And what happens to the animals?

    Nicola Reid (21:11):
    It's very important that we look after this magnificent creature. People say it's a living fossil, 250 million years old. It's survived a lot, so we need to make sure it continues to survive, it's a very important creature. So there's many things that are done, from a conservation perspective, not just from ourselves, from the other LAL vendors, as well. Everybody is very active in the conservation process for horseshoe crabs.

    Also, they're brought into the facility to be bled, to take blood from them. So if we just talk a little about that, I think is important, that they're hand collected by licensed local fishermen. They're hand collected, they're brought into the facility, and we take a small amount of blood from them, a small aliquot. We can't overbleed them, there's a protection in their system anyway, but we just take a small aliquot. They're then taken back and hand placed back again. It's a very fast process, we ensure we have them back within a maximum of 24 hours, it's usually much more quickly than that. And then that small aliquot, we move on then, obviously, to produce the LAL.

    And with the cartridge technology that we were discussing earlier, we need less and less of that. So we don't need to bleed quite as much because we don't need as much on that cartridge technology, so it really is a step forward. Not just these are the programs or the aquaculture with the protection of them, but also for us to look at methods and ways to reduce the amount needed but still give that good test, that regulated test that we know works very well.

    Norm Wainwright (22:52):
    Yeah, Nicola, I think the other aspect of this conservation is that there really has been, historically, a very bad treatment of horseshoe crabs going back to the earliest part of the 20th century and before where crabs were harvested in huge numbers and used for fertilizer and agricultural industries. I think overfishing for that purpose had a dramatic impact on the population, as well as the natural habitat for the horseshoe crab has become under more and more pressure, especially in the northeast where the human population has moved closer to the shore and populated many of those areas which used to be pristine estuaries and breeding grounds for the horseshoe crab.

    Gina (23:46):
    So what is the rate of mortality for biomedical purposes? Because, Norm, what you're describing, that sounds like 100% mortality if they're being used for that purpose. What do you see as mortality from the biomedical use?

    Norm Wainwright (24:04):
    I think it's safe to say that everyone collecting the horseshoe crabs in the industry takes good care of the animals, and I think there's certainly self interest involved where we depend on them for a production source, so naturally, we would do the best we can to protect them. But there are losses when crabs are handled and just taken out of the water, transported to the bleeding operation, then transported back. So there is an estimate in the industry that there's about 15% loss in this process. We in South Carolina are watched very closely by the Department of Natural Resources and our estimates of losses are somewhat better than that, somewhere between 6% or 8%. But even so, the amount of loss from the collection and return of the horseshoe crabs is minimal, I think, compared to crabs being collected to use for bait, which is 100% mortality, or in the old days, taking huge numbers and basically going to the fertilizing.

    Nicola Reid (25:23):
    Yeah, and that's clear in the ASMFC stock report, as well, that they've states, "It's a small number that we take for biomedical purposes in comparison to what they take for the bait industry anyway." And then the estimated mortality on that is smaller again. So it's almost negligible. They do say it's a minuscule amount in the report, not something to be concerned about, not something that would negatively impact the population. There are a number of things listed that could or would negatively impact the population - baiting, because we say it's 100% mortality. But now, there are restrictions on the numbers that they can take and that is getting better. The more preservation orders in place, the less baiting collection there is for bait.

    But, as Norm described, loss of habitat, various different environmental conditions, can also impact the horseshoe crab and therefore, that impacts further ecosystem. As Norm said, there are birds that feed on this, one of those being the red knots. So quite often, people concerned that our biomedical industry directly involved with reducing the number of horseshoe crabs, therefore reducing the number of eggs, therefore reducing the number of red knots. And I think all the data that is out there, the independent data, clearly shows that that is not the case. The biggest concern here is overfishing for bait and from loss of habitat due to destruction of coastal waterways or environmental impacts. There's many of us work together and there's many other independent agencies that work to try and prevent that, as well. But in the reports, the numbers are very high. We're talking millions of horseshoe crabs here. When you look at other endangered species like tigers, etc, they're in the very low numbers, below 100, and we're talking millions. So we're in a very good place with the horseshoe crab and we intend to continue to reduce the amount that we need to use for testing whilst keeping that good sensitivity that we need attest.

    Chris (27:25):
    As part of her role in product R&D, Nicola has been involved in not only developing better ways to use her supply of LAL responsibly, as with cartridge-based LAL testing, but she and her team are actively seeking viable alternatives.

    In our next segment, Nicola and Norm unpack the development and progress of synthetic products, or recombinant proteins, which have been around for a long time, but still have not succeeded in achieving industry-wide adoption. Unfortunately, as you'll hear, there are many challenges to recreating naturally occurring biological products. But we get closer to the real deal every day. Chris (28:02): With that, I'll turn it back over to Gina.

    Gina (28:06):
    I'm wondering if we could now get into the topic of a chemically synthesized alternative to the LAL test that we've been talking about? I'd love to hear your perspectives. We keep hearing all this buzz around synthetic LAL, or what the industry calls recombinant technology. So, Norm. What can you tell us about recombinant LAL?

    Norm Wainwright (28:28):
    Well, I think it was very attractive from the early days of recombinant DNA technology that this might be able to be applied in this way. The gene that would code for each of those enzymes in the enzyme cascade in the limulus cells, those genes can be engineered by placing them into another cell - a bacterial cell, a yeast cell, even an insect cell or a human cell - and that gene would then be read in those cells in culture so that the enzymes could be produced synthetically in the laboratory rather than being collected from the animal.

    However, the situation is a little more complex than a single gene. It really takes all three of those enzymes acting sequentially so that factor C is activated by the bacterial endotoxin, it then activates the second enzyme. And then, the second enzyme activates the third enzyme. Each of those steps gives amplification to the signal of the endotoxin binding to the first enzyme so that, in the end, there is the ability to detect extremely small amounts of material that gets amplified by this method.

    So when we talk about recombinant technology replacing the test, and Nicola mentioned the factor C as the first enzyme, it was used by itself. That is, one enzyme, not the full cascade. There's a difference, then, in how that reaction could be reacting to the endotoxin, with the possibility that it would not be as good, say, as the natural product in picking out endotoxin contamination that would be naturally occurring.

    Nicola Reid (30:35):
    Yeah, that's correct. We did some studies on this, we've recently published a paper, also. We produced our fully recombinants, we call it recombinant LAL, which was the three proteins, the three recombinant proteins. We compared that to standard LAL, biological LAL, and we also looked through a number of the commercially available RFCs so that we could see the performance of each of them and how they compared. We were really focused on what we call non-inferiority. We collected a number of water samples from our pharmaceutical customers globally. Samples that were a part of the WFI pretreatments and we knew what contained endotoxins because we wanted to challenge this with what we call natural environmental endotoxins.

    What we actually found in the study is that, in comparison to LAL - which is what we use now, which what is standardized globally - the RFC underpredicts endotoxin quite heavily. The recombinant LAL also underpredicts, not quite as much as the RFC does, but still does underpredict. So we were in a position after that study that we have all three enzymes, but it's still not equivalent. It's still not the same as LAL. So that's kind of where we are right now as a company with Charles River, is that we have this really robust data that shows recombinant technology right now is not as good as LAL. It's not equivalent to LAL when we're looking at environmental endotoxins. Can we get there? Yes, I believe we can. Norm can detail that there's a lot of other things going on in that horseshoe crab blood, there's other intercellular proteins, other activity within that horseshoe crab blood that we are learning about, understanding, and perhaps we need to know a little bit more about those two to help us enhance this recombinant technology.

    But I do believe we will have a future of recombinant, we'll be able to move away from using less and less LAL, helping then to conserve the horseshoe crab further, and moving towards recombinant in the future. But it has to be right, it has to be perfect. We rely on LAL now for everything, for anything that's injectable or a medical device, an implantable, to make sure it's free of endotoxins. So if we're sick or we're in hospital and we need an IV of some kind that it's not going to make us more sick if there's endotoxin in there. So we need to make sure that anything that will take over in the future and take the place of LAL is as good as LAL.

    Norm Wainwright (33:27):
    I think we should remind ourselves that the horseshoe crab species have been around for over 450 million years, so they've had a while to evolve this rather complex mechanism to ensure that their blood cells could react with the broadest possible range of bacteria out there to be part of their immune system. We're playing catch up, in a way, but we're learning very quickly. As Nicola said, the formulation of the recombinant enzymes that we now have can be adjusted. We can perhaps tweak things to be able to get the performance of our synthetic LAL as good, or maybe even better than the natural LAL.

    Nicola Reid (34:25):
    Yeah. As I've said before, these are living fossils, 250 million years in the making. We're trying to create a synthetic that matches what they have in their circulatory systems. It's clever science, it's very, very, very interesting. I'm certainly finding it interesting and to see the progression of this is quite amazing.

    Gina (34:46):
    Yeah, it really is.

    Norm Wainwright (34:48):
    I just want to make sure, Nicola, it's 450 million years.

    Nicola Reid (34:51):
    Oh, sorry!

    Norm Wainwright (34:51):
    You said 250.

    Nicola Reid (34:51):
    Oh, 450.

    Norm Wainwright (34:52):
    It's a long time.

    Nicola Reid (34:52):
    A long time.

    Gina (34:57):
    So, Nicola, I know there was a recent regulatory decision made about RFC, what can you tell us about that?

    Nicola Reid (35:03):
    Yes, certainly. So recently in Europe, the EDQM, which is the regulatory body for the European Pharmacopeia, produced a new chapter, Chapter 2.6.32, which is for the use of recombinant factor C. That is actually a compendial chapter within the European Pharmacopeia that was published in January of this year, 2021. So we do now have a compendial chapter in Europe, but what's significant about this in, actually the EDQM presented on this, a lady called Emmanuelle Charton, she presented that. It's like an empty shell concept. We have this compendial chapter, however all products fall within monographs within the pharmacopeia, and also within the general chapter. And each of the monographs and the general chapters specify which method should be used for bacterial endotoxin testing, and all of those chapters actually specify LAL at the moment.

    So even though there's a compendial chapter for RFC, when you're following the monograph it guides you to utilize LAL. The position now for pharmaceutical manufacturers, if they're following the European Pharmacopeia, is that they can use RFC, but they would need to do a full alternative method validation because they wouldn't be using what is recommended in the monograph.

    So there is progression and movement within the European Pharmacopeia. With regards to the rest of the pharmacopeias globally, the three that we have which are harmonized for bacterial endotoxin testing are the European, which we just discussed, the U.S Pharmacopeia, and the Japanese Pharmacopeia. Both the U.S Pharmacopeia and Japanese Pharmacopeia have road chapters for the use of recombinant reagents. Theirs is a little bit more general to recombinant reagents rather than specific to RFC, as the EP have done. And they've been through the process of reviews and feedback and they're currently looking at those and making a decision on whether they wish to publish a specific chapter in the way that the European Pharmacopeia has.

    However, all of the pharmacopeias, including the Chinese Pharmacopeia, do recognize recombinant as an alternative method. So people could use it, but they would have to go through a full alternative method validation and then submit that to their local authorities for acceptance, as they would be deviating away from the normal LAL or TAL methodologies. There's been some movement but, on the whole, it's a very similar position as we have been for a while, that it still remains much of an alternative method, but then there's a compendial chapter in Europe.

    Chris (37:54):
    Throughout this episode, our guests have discussed how LAL testing must conform to regulatory guidelines. I think we're all familiar with how medical treatments must satisfy regulatory requirements. But these measures also apply to testing methods in manufacturing. In fact, microbial detection is a highly regulated component of good manufacturing practices, which ensure the safety of pharmaceutical products. You've heard how traditional Limulus amebocyte lysate testing is an approved compendial method. That is to say that it adheres to the standards set forth by pharmacopeia. Different markets are regulated by their country's pharmacopeia. U.S Pharmacopeia, or USP, as well as the European and the Chinese Pharmacopeia, and more. The compendial monographs that Nicola mentions define a set of standardized methods and specification testing for pharmaceutical raw materials in finished products. As a point of clarification, those who wish to adopt RFC-based testing of these products must complete the necessary validation for the testing of their specific products before results are accepted. And, of course, it all comes down to the accuracy of the results. The safety of the patients depend on it.

    Gina (39:05):
    So switching gears a bit, I know that vaccines are required to be tested for endotoxins as part of safety requirements. And with the surge of vaccine production for COVID protection, what does this increased testing mean to the population of horseshoe crabs?

    Nicola Reid (39:21):
    That's a really good question, and it's been one on many people's lips. There's been many reports on this. So actually, three major LAL manufacturers recently got together and I think we have a published statement, where they did the calculations based on, I think, the World Health Organization said it would need five billion vaccines globally in order to protect the globe from COVID. So what that means is, that doesn't mean we test five billion vials because these vials are made in large batches of maybe 500,000 vials, etc. And then you tend to test beginning, middle, and end of each of those batches. We did the calculations of, "Okay, how many batches may be made over time? And therefore, how many tests may be needed? And then, how much more LAL would we need in order to cope with this mass manufacturing that will occur?" And what was calculated was, it would be one extra day's production from the three LAL vendors in order to supply enough LAL for all the COVID vaccines, so although the number sounds dramatic, five billion vaccines, when it's actually calculated down to what is needed, it isn't a huge amount. And that is in the vial format as in the vial format of LAL.

    If we actually use cartridges, the cartridge technology, to test these vaccines, then it's even less LAL that we need. It's 95% less of that one day's production, so there are no concerns about shortages, about not having enough horseshoe crabs, or not having enough of the raw material. There is plenty of that available and we see that now. Vaccines are being manufactured, vaccines are being produced, and they're being rolled out globally and LAL supply has been consistent.

    Gina (41:16):
    Wow, I don't think I realized that we didn't need to test every single vial, but just a sample of the batches. That makes sense now, why people would be concerned with the LAL supply, but I think this really clears up the confusion.

    Norm Wainwright (41:29):
    I think it goes back to the connection that we all have with the horseshoe crab and I think we should realize that the horseshoe crab is really only one of many thousands of marine organisms that we have not explored yet. I think that there is just ample opportunity for the future. Who knows where the new products may be coming from? Such sources that the similar serendipities that occurred, that resulted in LAL, may well happen with organisms that we haven't even begun to study.

    Gina (42:06):
    The world of science, it's so fascinating.

    Well, thanks so much to you both. It was really interesting to hear your perspectives on this topic and I can't thank you enough. Thank you.

    Norm Wainwright (42:18):
    My pleasure.

    Chris (42:20):
    Thanks, Gina, Norm, and Nicola.

    This amazing biological asset certainly benefits us all. And it's good to know that the manufacturers have come together to ensure that our collective supply of LAL can meet the demand of testing the millions of doses. But it's also very interesting to hear about conservation efforts, and the work being done to explore recombinant technologies. I encourage our listeners to explore the data from the comparative studies Nicola mentioned, which you'll find in the show notes. As with any natural resource, it's our responsibility to use LAL wisely and investigate viable alternatives.

    So, as you can see, the horseshoe crab's contribution to the biomedical industry is essential to human health. The safety of every single injectable is dependent on this animal. It's our collective responsibility as humans to preserve and protect the natural resources that we share with this planet. Until there's a safe, proven, and reliable solution that is equivalent to the crab's remarkable blood, we will continue to fight for its protection.

    Next month, we begin a fascinating series about finding new uses for existing drugs. Join us in May to hear from Dr. David Fajgenbaum, a doctor, patient, and an advocate who found the treatment for his own disease in a drug intended for another purpose. You won't want to miss his inspiring story.

    That's all for now. Thanks for joining us at Vital Science.