The horseshoe crab has endured for over 450 million years. It has survived several mass extinctions including the one that killed off the dinosaurs. One reason for their incredible resiliency is their ability to fend off bacterial infection. Their blood contains cells that clot around invading bacteria, thereby protecting them from the attacking toxins. In this episode we talk with three experts about how this animal’s unique blue blood has become essential to modern medicine. We also talk about why horseshoe crab populations are dwindling, and what biotech is doing to address the shortfall.
The horseshoe crab has endured for over 450 million years. It has survived several mass extinctions including the one that killed off the dinosaurs. One reason for their incredible resiliency is their ability to fend off bacterial infection. Their blood contains cells that clot around invading bacteria, thereby protecting them from the attacking toxins.
In this episode we talk with three experts about how this animal’s unique blue blood has become essential to modern medicine. We also talk about why horseshoe crab populations are dwindling, and what biotech is doing to address the shortfall.
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Rachel King (00:58):
Welcome to the first episode of our fall 2023 season. As summer fades and those walks on the beach move into the rear-view mirror, perhaps your memories include strolling along the water's edge and coming upon a creature with a large, round, brown shell, the horseshoe crab. The horseshoe crab has endured for over 450 million years. It has survived several mass extinctions, including the one that killed off the dinosaurs. One reason for their incredible resiliency is their ability to fend off bacterial infection. Their blood contains cells that clot around invading bacteria, thereby protecting them from the attacking toxins.
(01:39):
Today, we talk about how this animal's unique blue blood has become essential to modern medicine. We also talk about why horseshoe crab populations are dwindling and what biotech is doing to address the shortfall. I'm Rachel King, and you're listening to I am BIO.
(01:56):
In the early 1960s, scientists discovered something remarkable about horseshoe crabs. Their blue blood was particularly sensitive to endotoxins, which it can detect and eliminate. When confronted by harmful bacteria, the crab's blood cells clot and surround the invading bacteria to keep it from causing any further damage. In humans, endotoxins can induce inflammation, fever, septic shock and even death, so it's vitally important that injectable pharmaceuticals, like vaccines or IVs, or medical devices like catheters or pacemakers, are free from endotoxin contamination.
(02:54):
Enter the horseshoe crab. Researchers discovered that the same clotting response of the crab to an invading endotoxin can be replicated in testing medical products. Samples are mixed with a reagent called LAL, which is made from the horseshoe crab blood. If a clot forms, the product is contaminated. The test is used in an array of pharmaceutical products and has helped keep most of us alive at one point or another. The good news is, most horseshoe crabs are returned alive to the sea once they are bled. The bad news? Excessive baiting and pressures from climate change have contributed to a decline in horseshoe crab populations, and the species is now considered threatened. Today, we take a deep dive into the miracle of the horseshoe crab and why fighting to sustain it is worth the effort.
(03:47):
Our first guest is from a large pharmaceutical company that tests tens of thousands of samples for endotoxin contamination.
Jay Bolden (03:55):
Hi, my name is Jay Bolden. I'm a director in the Global Quality Laboratories at Eli Lilly and Company in Indianapolis, Indiana in the United States.
Rachel King (04:04):
Jay talks to us about the history of using horseshoe crab blood in drug testing and how it has changed over the years.
Jay Bolden (04:10):
The unique properties of the horseshoe crab were discovered back in the 1950s and '60s by Frederik Bang and Jack Levin at Woods Hole. They were studying the blood system of the horseshoe crab. What they noticed was that in the presence of contaminated seawater that was contaminated with bacteria, they noticed that horseshoe crab's blood system clotted around that contamination. And from that observation was born the bacterial endotoxins test, which up to that point had been tested in rabbits.
(04:46):
So in the 1940s in the United States, the way to ensure the safety of your medicine was to inject a rabbit and then monitor it for fever. The rabbit pyrogen test itself, you would inject typically your final product into a rabbit, and you would monitor that rabbit over three hours for a fever. It's a qualitative readout. If the rabbit exceeds X amount of degrees, see increase in body temperature over that period of time, then your product is adulterated and you can't release that product to the market. But then if the rabbit does not have a fever that meant your product was pyrogen free, and then it was of suitable quality to release to the market.
(05:26):
The rabbit pyrogen test itself utilizes three rabbits and up to eight and that's just per test. So I think historically a lot of pharmaceutical companies had colonies of rabbits that had to be used for this kind of testing.
Rachel King (05:41):
Even with colonies of rabbits, it would be difficult to test tens of thousands of samples. It simply wouldn't be done. Jay explains the importance of the horseshoe crab blue blood discovery.
Jay Bolden (05:52):
The discovery of LAL, which is Limulus amebocyte lysate, it's the purified horseshoe crab blood. It really changed pharmaceutical manufacturing quality control in that the standard up until the 1980s was a full-on animal test. And so, the discovery of LAL and the subsequent implementation of that as a national standard test method that made obsolete the rabbit pyrogen test, but it also enabled the ability to test a lot more samples.
(06:24):
At Lilly, you know, we test tens of thousands of samples per year with an endotoxin test, but you wouldn't likely have done that with rabbit testing.
Rachel King (06:32):
Just describes the process for creating the test.
Jay Bolden (06:35):
The gist of it is that crabs are collected, a small amount of blood is taken from the crabs and they're generally returned back to the sea. That blood is further purified. The cells themselves that contain the protein that detect the bacterial endotoxins is lysed, and then it's formulated into a test reagent. You would take a glass test tube, you would put your sample in it, you would put an amount of this purified horseshoe crab blood, the LAL, in it. You would incubate it for 60 minutes, invert that test tube 180 degrees, and then observe it for a clot, you know, just like it would have clotted inside the crab or not. If it clots, it sticks in the glass tube and you're positive for endotoxin. If it falls out, you're negative for endotoxin.
Rachel King (07:21):
More from Jay in a bit, but now I'd like to introduce our second guest.
Deborah Cramer (07:26):
My name is Deborah Cramer, and I'm a science writer. I've written three books about the sea and the shore, and I write occasionally for the New York Times op-ed page.
Rachel King (07:37):
For Deborah, it's imperative that the horseshoe crab survives.
Deborah Cramer (07:41):
Protecting and rebuilding the horseshoe crab populations in the United States is essential. Horseshoe crabs are a limited supply, and the demand for this test is virtually unlimited as the pharmaceutical industry continues to expand, as personal medicine continues to increase, because if you have personal chemotherapy or other immunotherapy treatments, the endotoxin test has to occur on all of that. So the demand is virtually unlimited.
Rachel King (08:16):
Deborah describes when she realized there was a problem with the horseshoe crab population and why we needed to reverse the decline.
Deborah Cramer (08:23):
I live at the edge of a tidal creek and many, many years ago, I used to see horseshoe crabs coming into my creek to lay their eggs. And I was thrilled and excited to see these animals that are so old come into the creek. And then, they disappeared and I wanted to know more about why they disappeared and who they were. And I learned that they've survived every one of Earth's mass extinctions and they seem to come from a entirely different realm. If you're on the beach in the middle of the night, half an hour before high tide, they come in, thousands of them, to lay their eggs and then they disappear in the darkness half an hour later and it's as if they've never even been there.
(09:12):
The number of horseshoe crabs spawning in Massachusetts, for example, where I live, hasn't substantially or meaningfully increased in years. Used to be able to see lots of horseshoe crabs spawning on beaches in Massachusetts, it just doesn't happen that much anymore. The horseshoe crab populations in Long Island and New York are in poor condition. The populations in Delaware Bay are stable. They're not continuing to decline, but they're not increasing either. And in South Carolina, where we had all believed for many years that the population was stable or growing, in the past few years, the spawning of horseshoe crabs on beaches has also substantially declined.
(09:59):
I'd say there's an ethical imperative not to allow an animal that has existed for so long to disappear. The horseshoe crab is a, a species on which many other animals depend, and as those horseshoe crab numbers decline, those other species, particularly the birds, are not managing very well.
Rachel King (10:26):
Deborah's interest in and knowledge of the horseshoe crab stems from her research on a tiny endangered shore bird called the red knot, something she has written about in her book, The Narrow Edge.
Deborah Cramer (10:38):
The red knot is listed as threatened under the US Fish and Wildlife Service Endangered Species Act. It's listed as endangered in a number of other countries. I was really fortunate when I was researching and writing The Narrow Edge that I was able to accompany the red knot from its winter home in Tierra del Fuego at the bottom of South America all the way up into the Arctic where it lays its eggs.
(11:03):
This is one of the Earth's longest bird migrations. And along the way, the birds need safe passage in a place where they can get all the food they need to make the next leg of their trip. When they arrive in Delaware Bay, for example, from Northern Brazil, they need to double their weight to make the flight to the Arctic. And they double their weight eating horseshoe crab eggs. There needs to be enough horseshoe crabs on the beach so that the crabs are so dense when they come into spawn that they overturn each other's nests and eggs come to the surface. Because the bill of the red knot is not long enough to go into the sand and pull out the eggs.
(11:51):
Every red knot that lands in Delaware Bay needs to eat 400,000 eggs in 10 days to gain the necessary weight to breed successfully in the Arctic. There used to be maybe 150,000 red knots down there. That's a lot of eggs to restore the population of red knots. And there are many other birds that aren't yet on the endangered species list, but that are beginning to qualify, that also eat and rely on horseshoe crab eggs.
Rachel King (12:23):
While some might argue that one little bird doesn't matter, Deborah has a different view.
Deborah Cramer (12:28):
In The Narrow Edge, I wrote a whole chapter about this called Does Losing One More Bird Really Matter? And lots of times, a species disappears and it's only after it's disappearing that we actually realize what it's doing for us. We don't know, we can't quantify right now the economic value of a red knot. I would argue that we've made so many mistakes in the past, letting species disappear and then discovering their value to us, that we can't really afford to take that kind of risk losing so many shorebirds.
(13:10):
Shorebirds play lots of roles in the environment. They carry seeds from one place to another, dispersing them. For example, after huge volcanic eruptions that basically scour a place, it's birds that often bring in the seeds that bring about the regrowth. I can't quantify for you the value of a red knot. I would say though that I feel uncomfortable with our species making decisions about who can live and who can die based on our perceived understanding of their value to us. These animals have the right to exist in and of themselves, especially when there's no reason that we have to do this. We have a huge alternative to taking horseshoe crabs to make endotoxin tests, and we have no need to take horseshoe crabs for bait, so we can provide thousands of shorebirds with the food they need.
Rachel King (14:21):
When we come back from a break, we'll talk about the alternative endotoxin test that Deborah just mentioned. For the first time since 2019, we're hosting the Bio Investor Forum in person. Join us at the Hilton San Francisco Union Square from October 17th to the 18th. You'll experience two days of thought leadership, bio one-on-one partnering, and insight into investment trends for early stage biotech companies. Visit bio.org/events.
(15:14):
Of course, we're grateful for the incredible contribution the horseshoe crab has made to ensuring drug delivery is safe and free from contamination. But as Deborah pointed out, this is a finite supply. Current estimates put the number of endotoxin tests performed per year using horseshoe crab blood at over 70 million. That number will only increase as drug development and medicine continue to advance at a rapid pace. Our third guest is from a large biotech company that is transitioning to a synthetic endotoxin test.
Lindsey Silva (15:47):
My name is Lindsey Silva. I am a Director of Microbiology and Analytical Sciences, Pharma Technical Quality and Compliance at Genentech, a member of the Roche Group.
Rachel King (15:59):
Lindsey describes the new test for us.
Lindsey Silva (16:02):
The good news is that there are now additional synthetic reagents on the market such as recombinant factor C and recombinant cascade reagent that can be used for endotoxin testing that do not rely on horseshoe crab blood. There is nothing wrong with using a compendial method, such as the endotoxin test method that relies on horseshoe crab blood or the synthetic method, such as recombinant factor C.
(16:32):
So the rFC method includes only a single recombinant enzyme factor C. So when endotoxin comes into contact with recombinant factor C, it initiates enzymatic reactions, resulting in the detection of the fluorescence output. So the benefits of recombinant factor C adoption is that it's a reliable assay in terms of improving specificity for endotoxin testing.
Rachel King (16:59):
Genentech has been evaluating rFC for some time and is convinced that it is reliable and safe.
Lindsey Silva (17:06):
So when we are considering reliability of a test method, we need to evaluate that it is fit for its intended use and safe for patients based on science and data. Our scientists and engineers have spent much of the last two years evaluating, validating, and implementing an endotoxin test method for pharmaceutical water samples using recombinant factor C.
(17:34):
So after a proof-of-concept study in South San Francisco, we conducted pilot studies in Kaiseraugst, Switzerland. So far, we have data demonstrating that recombinant factor C is reliable, safe, and can replace LAL testing as a standard for endotoxin testing in the pharmaceutical industry. At this point, we are performing water testing at our Kaiseraugst quality control manufacturing site using the recombinant factor C method, and we are taking the next steps to evaluate the suitability of using recombinant factor C for injectable medicine testing.
Rachel King (18:15):
For Eli Lilly's Jay Bolden, his drive to find alternatives to blue blood was informed by his hobby and passion for bird watching.
Jay Bolden (18:23):
I've kind of been interested in birds for some time, but I started bird watching as a hobby in 2000. Incidentally, that's when I started at Lilly. I've been to all corners of the country at all different times of the year. That's what I like to do. So I've been doing that for 23 years at this point.
(18:39):
When I first started at Lilly, my job was to develop endotoxin tests. And at the time when I first started, I was told that someday we may not have access to the crabs. During the 1990s, crab populations had gone way down. But they also noticed that some bird populations went way down. And so, it turns out that there are migratory shorebirds that travel from the southern tip of South America or, you know, other places in South America all the way up to the Arctic and back in one year. A lot of them time their migration to hit the East Coast of the United States during crab spawning, which is April, May, June time frame.
(19:19):
Crab eggs, as it turns out, are a great source of protein and fat and so these birds fly non-stop. They're emaciated by the time they get here and they eat the horseshoe crab eggs and they go on and try to breed in Arctic. As the crab populations went way down in the 1990s, so did one particular bird called the red knot, and that was known to me in bird-watching circles. I very much knew that our test reagent came from horseshoe crabs, and I understood the dynamic between horseshoe crabs and red knots.
(19:51):
And so, we had looked at a synthetic alternative to LAL, which had come onto the market in 2003. It was patented in 1997 by Ding Hong Ho at the University of Singapore. At Lilly, we had looked at this reagent called rFC, which is just the synthetic version of the horseshoe crab protein that detects endotoxins in 2006. And we passed on it for various reasons, including there was a lack of regulatory guidance. There was only one supplier, so there was kind of a supply chain concern at that time. In some ways that the intersection of birdwatching and my professional interest, it's great that I can have an impact on something through my work.
Rachel King (20:31):
And Eli Lilly began its move toward a recombinant test 10 years ago, as Jay explains.
Jay Bolden (20:37):
A lot of things changed in 2013. The FDA came out with endotoxin testing guideline in 2012 that said how they would accept rFC. Importantly, a second supplier entered the market, so now you wouldn't have all your eggs in one basket, as it were. The US Fish and Wildlife Service listed the red knot as threatened, so it was a, you know, migratory shorebird whose populations had gone way down. And we were concerned about impacts to horseshoe crabs if the red knot was listed as endangered.
(21:09):
And we also were aware that there are only two places to find horseshoe crabs in the world, and that's on the eastern coasts of North America and then the eastern coasts of Asia. I had been to presentations, I had seen concerns about populations of horseshoe crabs in Asia and they were doing much worse than in the United States. So in 2013, Lilly started building a new plant in China and I was concerned about supply chain and our ability to get the test reagent there. So a lot of factors came together in 2013 that made us think we should start looking at rFC again.
Rachel King (21:48):
Jay helps us understand why taking the animal piece out of the test is such an exciting development.
Jay Bolden (21:54):
The synthetic alternative to LAL, recombinant factor C, is a product of biotechnology. Biotechnology itself is a 40-plus-year-old technology. Eli Lilly was the first company to introduce a recombinant medicine to the market instead of getting insulin from pigs and cows. It's made in cells now, and we've been doing that for 40 years. So recombinant factor C is simply the DNA that creates the horseshoe crab protein that detects endotoxins and it's created in cells. That DNA sequence is created in cells. It's grown and it's purified. So it completely takes the animal piece out of the question.
(22:35):
One of the benefits of rFC produced from biotechnology is that it's theoretically an infinite resource. And compare that to horseshoe crabs, which are a finite resource. It's a finite natural resource. They're not farmed, they're taken from nature, and clearly their populations have gone down and they're not appreciably coming back up. The ability to make a synthetic version of that important protein that protects human health, to protect the supply chain in a way that's much more sustainable than LAL, that's really important. And I do think there is momentum in industry.
Rachel King (23:10):
And Eli Lilly has successfully launched products using the new test.
Jay Bolden (23:14):
We made the commitment in 2016 after, you know, generating data for several years that we would convert our water testing and any new products and we would start using rFC to release those instead of LAL. We had a first in industry product approval in 2018. It was the first product that we tested using rFC and released to the market. We submitted that to the FDA. The FDA looked at our data and they agreed with us that rFC was equivalent to LAL. And so, since then, we've gotten at least seven more products approved and we've gotten approvals all over the world in at least 60 different countries, even for that first product.
(23:51):
I'm happy to see that some of our industry peers, including Sanofi and Pfizer and Roche are doing something very similar to what we've done in that they've committed to converting water testing, which makes up a huge volume of endotoxin testing, and that new products that they're going to attempt to use rFC for their new products as well.
Rachel King (24:10):
Lindsey says that, indeed, Genentech Roche is committed to transitioning to rFC.
Lindsey Silva (24:16):
We are focused on implementing recombinant factor C as an alternative non-pharmacopoeial method. We are prioritizing recombinant factor C application for water testing due to the low regulatory impact. In addition, pharmaceutical water samples represent 80% of the endotoxin samples tested per year in the Roche network. By making the switch from the LAL assay to recombinant factor C for water testing, we are already observing an impact on reducing our reliability on using horseshoe crab derived-material as an endotoxin test method.
Rachel King (25:03):
Lindsey and Jay agree that if there is a viable means to protect both ourselves and the animal ecosystem, we should use it.
Lindsey Silva (25:11):
Respect for a healthy planet and the environment has always been a priority for Roche and Genentech. We certainly hope that adoption of recombinant factor C will have a positive impact on the horseshoe crab population, because we realized that reliance on the Asian horseshoe crab population for a quality control assay is not sustainable. Our mission is to deliver life-saving medicines for patients. We want to make sure that when we test for the safety of our medicines, that we are doing it in a sustainable manner. One step in the right direction is to ensure that we are using test methods that do not rely on animal-derived material.
Jay Bolden (26:04):
Horseshoe crabs have protected patient safety for decades, but there's a better way to do it now. And so, caring about, not just the horseshoe crabs, but all the species that depend on horseshoe crabs, it's not just birds. Turtles eat horseshoe crabs, horseshoe crabs eat other things. It's an ecosystem thing, you know, everything's connected. And so, when you impact one thing, it, it not only impacts that one thing, it impacts a lot of things.
Rachel King (26:31):
The miracle of the horseshoe crab's blue blood has made modern medicine possible and saved lives. Now biotechnology is returning the favor by reproducing this resilient animal superpower with a recombinant version, hopefully allowing the horseshoe crab to endure for another 450 million years. I want to thank our guests, Jay, Deborah, and Lindsey, for helping us understand that our environmental ecosystem thrives only if all of its components thrive as well. And thank you all for listening.
(27:04):
Make sure to subscribe, rate, and/or review this podcast, and follow us on Twitter, Facebook, and Instagram at I Am Biotech. And subscribe to Good Day Bio at bio.org/Good Day. This episode was developed by Executive Producer Theresa Brady, and producers, Lynne Finnerty and Courtney Gastinelle. It was engineered and mixed by Jay Goodman with theme music created by Luke Smith and Sam Brady.