August 21, 2021

The old biotech and the sea

By Conor McKechnie and Dodi Axelson

The old biotech and the sea

Short and sweet - Conor brings an interesting story about role of horseshoe crabs in the pharmaceutical industry. Guest and subject matter expert is Ding Jeak Ling - or Lynne, as she prefers to be called. She is a professor at the Department of Biological Sciences National University of Singapore, and her main research interest is in Innate immunity and cancer immunomodulation.

CONOR: So, Dodi, you know how we stumbled across all sorts of like weird and wonderful aspects of science as we learn about our industry, and we're finding that biotech relies on all sorts of like incredible accidental discoveries in nature, and these get turned into tools and technologies at an industrial scale?

DODI: The weirder the better. But an example, I guess, of what you're talking about is protein A, the workhorse of so many biologic purification processes that are being discovered as a surface protein on Staphylococcus aureus bacteria, for example.

CONOR: The episode that kicked off all of this. Yes, absolutely. And we talk about the fact that, you know, our industry is really innovative, but it's also really conservative.

DODI: Yeah, there are new discoveries all the time. And once tried and tested, methods get proven and are shown to be safe, that can be hard to change, even if those changes mean improved efficiency, reduced costs, and even better safety.

CONOR: Yeah, it's like a double-edged sword. So patient safety is paramount. So, processes are really, really strictly regulated. But improving drug manufacturing processes could make them better, could make them faster and more sustainable.

DODI: And I guess all of that is what matters in today's episode of Discovery Matters.

CONOR: So, what if I told you Dodi that something that we rely on as fundamental to patient safety across the whole of the pharmaceutical and medical technology industry is 450 million years old? Older than dinosaurs? Impossible? Okay, I'm going to send you a picture. Are you ready? One, two, here you go.

DODI: So that looks like, I don't know some Star Trek™ alien life force spitting out?

CONOR: Isn’t it crazy? So, these are horseshoe crabs, and they are being milked for their blood, which is hypersensitive to toxins from bacteria. And pharmaceutical and med-tech companies use that blood to test things that are injected or inserted into our bodies for contamination. It's basically to stop patients dying, right so vaccines, medicines, stents, pacemakers, anything that goes into our bodies, all the batches need to be tested for something called endotoxins before they can be shipped, and then used in humans.

DODI: So that sounds heroic, but it's probably not so nice for crabs. But to be current and to be newsworthy, are we also including COVID vaccines in this test?

CONOR: Absolutely. And you can imagine how much crab blood is going to be needed if we're going to vaccinate a planet's worth of people, right?

DING JEAK LING: You know, the horseshoe crab is really such an endearing animal.

DODI: Alright, introduce us, who's that?

CONOR: This is Professor Ding Jeak Ling at the National University of Singapore. She goes by Lynne.

LYNNE: I was a kid when my dad brought home a horseshoe crab, he went fishing, and he brought one home. And I was really amazed by something that looked almost like an alien thing from outer space. And what does it do? I didn't even know where the head or the tail was, and it was totally harmless. It was a God-given gift to humanity. So why do we let it become extinct? Many lessons about animals have gone extinct.

CONOR: But before we feel bad about the crabs it used to be worse, it was rabbits.

DODI: Not Peter Rabbit.

CONOR: Yeah, those little bunnies. So, you know, from the 1940s to the late 70s. The rabbit pyrogen test was the main way of ensuring human safety from bacterial toxins in medicines. The rabbits were injected with test material and then they were monitored for temperature rises.

LYNNE: So, the endotoxin detection in surgical implants have, since 1942, used the rabbit pyrogen test. But we know rabbit pyrogen test is very expensive. It is not user friendly. It is not environmentally friendly, not any more friendly, and it has drawbacks because of the variations in sensitivity to endotoxin detection. It is slow, takes a few hours to know the result and very prone to false positive or false negative.

CONOR: Lynn's really modest but actually she is a huge deal.

DODI: Did you get to be a fanboy again?

CONOR: I know I did. The rabbit pyrogen test was replaced by what's called the LAL test or the limulus amebocyte lysate test in the late 70s. It's the LAL test that depends on this gorgeous beautiful blue blood of this amazing horseshoe crab.

LYNNE: Limulus amebocyte lysate is derived from the blood of the horseshoe crab. And as and when you get the horseshoe crab from different seasons of the year, or different places in the world, that there will be batch variations. Different methodologies of preparing the LAL would also cause differences in the endotoxin detection capabilities. And the horseshoe crab itself is already very highly endangered.

CONOR: About 500,000 horseshoe crabs die every year during this process.

LYNNE: And a good percentage of these horseshoe crabs will also die during this process. So, you see the blood is blue, because it contains hemocyanin copper, unlike our red blood, which contains iron, so they also carry oxygen, but in these blue tubes of blood, see the horseshoe crabs are live, they are bent, curved pieces bent and there is a little septum here, which the producer will stick a needle in. So, it will be partially bled.

DODI: So, save the rabbits but doom the crabs.

CONOR: Pretty much. And it's the LAL test that Lynn has been working on for almost 20 years to replace, not just to save the crabs, because the aim is to develop a test, or she has developed a test that is better at detecting endotoxins than the standard LAL test. So, you know, we have this recurring theme of happy accidents and scientific discovery?

DODI: And the word serendipity, we love it.

CONOR: Lynne didn't set out to change endotoxin testing at all. That's not what she was doing. She was trying to solve a problem for Singapore's test-tube baby programme.

LYNNE: But we were driven. We were very driven. Because to start with, we were made to become 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 that you can measure something like 100 samples or less with because you need to make your calibration curve. And in Singapore, in the mid-80s or late 80s, there was a group of clinician scientists who were helping the test tube baby programme and the human embryos were becoming contaminated. They were dying prematurely. So, they suspected there was some bacteria. And they asked my colleague to measure/detect the presence of gram-negative bacteria. So that was the starting point, he could only afford to bring in one kit and we had no money. It was early days, you know, 1980-something, was really quite primitive.

CONOR: And so out of all of this...

LYNNE: A very simple, rapid and standardized non-animal derived Recombinant Factor C (RFC) was engineered for detection of endotoxin.

DODI: Well, does it work?

LYNNE: Actually, the RFC is very well received, extremely well received by various groups of people. Firstly, of course, conservationists, they want to ensure that the horseshoe crab will not die off. Secondly, and very importantly, the biomedical sector industry. I think Eli Lilly has over five years, exploring the use of RFC, they have tested 16,000 samples using RFC and in direct comparison of these 60,000 samples, RFC on one hand, and LAL on the other hand, direct comparison, and found that the RFC was efficacious in detecting endotoxin, if not more specific, and does not suffer from false positives.

CONOR: So, the industry is changing, but it's really slow. And it does require action by regulators to recognize the new test as equivalent or better. And then to update the regulation, and then drive uptake.

DODI: And if there's anything that we've learned during this pandemic year plus is that the regulators can move faster.

CONOR: And they do and this is something that we really hope, you know, continues post-pandemic as we make the system more efficient, and beneficial. So, you're absolutely right, regulation has increased in its speed to deal with a pandemic. And of course, we're hoping that this becomes a permanent way of getting medicines to market faster.

LYNNE: There is something like 50 or so drugs produced by Eli Lilly through the use of Recombinant Factors C, and something make more than 10 I'm not sure it keeps increasing, of these drugs have been approved by FDA.

DODI: So, this is a call to the industry to save the crabs, really, to keep this very important process of testing for endotoxins. But let's save the crabs because the world probably needs these crabs.

CONOR: And you know, the coolest thing about the horseshoe crab?

DODI: I bet you're going to tell me.

CONOR: It's not even a crab. It's actually more of an insect. So, there you go. And it is millions of years old. So, these living fossils...

DODI: 450 million you said...

CONOR: Indeed, and we want to keep them around because they're just cool!

DODI: All right, here's to the cool crabs of the world, and if you're a cool crab then give us a rating, please!

CONOR: So yeah, crabs for the win and our executive producer is Andrea Kilin. Discovery Matters is produced in collaboration with Soundtelling. Production by Tanvir Mansur. Our theme song was written by Thomas Henley and additional music is from Epidemic Sound.

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