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Sounds of Science is a monthly podcast about beginnings: how a molecule becomes a drug, how a rodent elucidates a disease pathway, how a horseshoe crab morphs into an infection fighter. The podcast is produced by Eureka, the scientific blog of Charles River, a contract research organization for drug discovery and development. Tune in and begin the journey.
Mary:
I am Mary Parker and welcome to this episode of Eureka Sounds of Science. Today I am joined by a couple of prominent experts in the field of limulus amebocyte lysate, or LAL testing. Norm Wainwright and Foster Jordan are both retired from Charles River and in their long careers they have seen and had a part in the remarkable evolution of pyrogen and endotoxin testing. This year marks the 20th anniversary of the Endosafe cartridge technology invented at Charles River to streamline LAL testing, so what better time to discuss the history of how we keep patients safe from harmful bacteria. Thank you for joining me, Norm and Foster.
Norm Wainwright:
Glad to be here.
Foster Jordan:
Yeah, thank you for having us, Mary.
Mary:
Glad to have you here. Foster, welcome back. I know you've been on here before and I appreciate that.
Foster Jordan:
Thanks for having me again.
Mary:
So can we start with your backgrounds to set the stage. Norm, how did you get started in this field?
Norm Wainwright:
Well, before I started with Charles River, I was a scientist at the Marine Biological Lab in Woods Hole, Massachusetts.
Mary:
Oh, cool.
Norm Wainwright:
Of course that is where the LEL cascade was originally discovered by two scientists at Levin and Bang. I had some experience with LEL. I also had some experience with primitive immune systems of other marine invertebrates. It was a natural, when I did start with Charles River doing R&D with Foster on a number of small projects, I was really an external source, working in Woods Hole remotely. Eventually, I moved the lab entirely to Charleston and stayed there ever since.
Mary:
This is a tangent, but were there any particularly cool marine invertebrates that you enjoyed studying while you were at Woods Hole? I love marine biology.
Norm Wainwright:
Well, it's funny because almost any marine organism is unique in its own way, and that's really the beauty of what was happening in Woods Hole. The purpose of the whole institution was really to take advantage and exploit in the best possible way, all of the different diversity of biological mechanisms that exist in the marine world. The challenges are so great, it's a very harsh environment, and so a lot of the solutions to biological questions, biological problems that an organism may face, whether it be defense against microbes, as in the case of the horseshoe crab, or defense against other marine organisms, the biological molecules that were created throughout evolution to cope with those things are really fascinating and a great source of new biomedical type of products.
Mary:
Yeah, you bring up a good point. We do have it pretty cushy here on dry land. How about you, Foster? What's your experience in this field?
Foster Jordan:
Dr. Cooper hired me right out of graduate school to do R&D projects around quantitative detection of bacterial endotoxin in 1991. I worked in R&D through 1994 when Charles River acquired Endosafe. Between 1994 and 1997, as Dr. Cooper phased out and began to retire, I assumed more management responsibility. Then in 1997, I became the business leader for Charles River for the Endosafe division at the time. Then over time it evolved into Microbial Solutions as we added more products and added more acquisitions. I've been running, I've been the business leader for the Microbial Solutions division as it is today, since 1997, and I retired last year, but at the time when I retired, we had obviously the entire branch were doing Endosafe, [inaudible 00:04:20] and Accugenix, so I was running that division.
Mary:
All right. We've talked a lot about LAL testing on the podcast, but just in case someone is hearing this for the first time, foster, can you explain what it is?
Foster Jordan:
Yeah, so limulus amebocyte lysate is a invitro diagnostic reagent that is used and FDA approved to detect for the absence of gram-negative bacterial endotoxin contamination in injectable drugs, medical devices, and other products that come in contact with human blood to prevent what is called as a pyrogenic reaction, which is when the body detects the bacterial contamination in the drug, you get a fever response, you could actually be so severe that it could ultimately cause death. It's required by the FDA to test for the absence of these bacterial endotoxin contaminations in all injectables and medical devices.
Mary:
This is the thing where the toxin could be introduced during the manufacturing process accidentally, that sort of thing?
Foster Jordan:
Yeah, whenever you have people in air and water, you have bacteria, right? Since you're high level, high amount of water involved in manufacturing, a lot of people involved in manufacturing, you have the opportunity to contaminate with bacterial or other microbes, and this is a way of screening all of your raw materials, screening all of your equipment, as well as a final product release test to make sure these bacterial contaminants are not present in the final product upon release.
Mary:
Perfect. We've said cartridge a few times now. Norm, can you briefly explain how the cartridge works and how it came to be?
Norm Wainwright:
Yes. The challenge with the LAL test is that it's in the conventional way that it's performed is a pretty demanding test. It takes good technical skills, it takes time to do the proper dilutions, preparations of standards, dissolution of all the reagents, and then reading the test with instrumentation or just manually reading a clotting reaction. For our customers, it was really a relatively expensive technology to engage in, not so much from the reagents, but rather the technician time to perform the test and do the analysis. The cartridge technology was really an answer to how we could streamline that a bit, make it more user-friendly, take out of the technician's hands, the need to do all of those demanding tasks and have them just concentrate on collecting the sample and putting it into a device and an instrument that could perform the test fluidics for the technician be controlled by a computer.
It could have a hardware in the form of pumps to move a sample from point to point. It could then move a sample to where it could be analyzed with a spectrophotometer, which is the way LAL test is typically performed. Side by side with Foster and a number of engineers and scientists, we developed a means to use microfluidics to dry the reagents for LAL in channels, and we put the positions of the reagents in the channel similar to how they would be formed manually. As a sample entered a channel, for instance, as a liquid, it would dissolve the dried reagents in the channel sequentially as it moved down the channel. Then at the end of the channel, after it picked up the LAL reagent, it incubated with the sample it mixed with a chromogenic substrate. It moved finally to a position where a miniature spectrophotometer could read the results of that test.
All of this was in the form of a handheld instrument. We really shrunk the volume of reagents that are needed for the test, removing the burden of the technician to perform all those tests. Having them done by the handheld reader was a pretty daunting task, but it's one that really made a big difference in terms of the time to perform the test and the energy to perform that test.
Mary:
Yeah. Less blood, less material, and less expertise is needed. I would imagine that also made it cheaper, not just with labor costs, but with the chemicals themselves.
Norm Wainwright:
Yeah, absolutely. Foster, I think you did pretty much an analysis on the percent of reagent that we ended up using and needing for the test.
Foster Jordan:
Yeah, it reduces the demand for our crude material by 95%. It's an amazing reduction on the raw material. But in addition to everything Norm talked about, it also for the first time ever, it decentralized the ability to do endotoxin testing. Historically, a sample was pulled and then logged into the QCLab, and then maybe the next day they analyzed it for production. But with our introduction to the PTS, the portable platform, the test could then move to the point of view.
You could actually pull a sample in real time, pull a sample on either a problematic area or an area where you needed a critical time to result, and that either anyone could run the test, even the production people could run the test to actually get a real quick number instead of waiting for the central lab to generate the test result, which was again, really helpful to pharmaceutical companies who have high risk samples during their in-process testing.
Mary:
Yeah. Well, and not just pharmaceutical companies, you reminded me that we did a video a couple of years ago about a kidney dialysis center basically in the middle of nowhere in Australia, very remote location, where a lot of aboriginal people lived and needed dialysis. Instead of having them move hours away to the next nearest city, they were able to stay put in their home village by the creation of a dialysis center. But that was only possible because they were able to test the water with the cartridge technology to make sure, because kidney dialysis water obviously has to be extremely pure. It helped patients in that way as well. Bringing this to remote locations. I'll add a link.
Foster Jordan:
Absolutely.
Mary:
I'll add a link to that.
Foster Jordan:
Similar to compounding pharmacies in hospitals, typically a compounding pharmacy would compound a particular custom drug for a particular patient, and that would be infused at risk, because there was no real way to do the endotoxin test at point of delivery. That also allowed for compounders to have the ability to do the test right before they injected the drug in the patient.
Mary:
Yeah. Okay. Yeah. We're going to start with some present issues and work our way back, but so recently, Charles River launched Endosafe Trillium, which is our own version of recombinant LAL that doesn't use horseshoe crab blood. That's a big step. Can you both tell me what the impetus was to create this product? Foster, you want to start?
Foster Jordan:
Yeah. I think everybody realizes that sustainability and conservation is critically important, and we realized that early that we needed to get involved even though it is a synthetic derived blood, but it's still the same sequences that the natural species use. We just use a mammalian cell culture, which Norm created to generate the three main proteins that are used to mimic the natural LAL test. But again, it's an evolution. We realized that we needed to do that. We were able to reduce the demand for horseshoe crab material dramatically by the introduction of the cartridge, but it was still important to know that this technology was coming and it would be the wave of the future, and we needed to get involved.
As the regulations evolve, obviously it'll continue to evolve and the recombinant will have a significant play in the field of endotoxin testing. Norm, as running the business, I reached out to Norm and said, "We want to divert a lot of your research R&D dollars to the development of recombinant material." Norm, you can talk through how that process worked and how you were able to develop Trillium.
Mary:
Yeah, let me pause there for a minute to give a little bit of background for anyone that doesn't know that basically there's a protein cascade in the blood, correct me if I'm wrong, that causes it to clot in the presence of an endotoxin. That clotting is what can we can use to alert us to the fact that there is a toxin in contact with the reagent?
Foster Jordan:
Well, because one of the things that I asked Norm to do is the reagents that were available or that were early in commercialization were single enzyme assays that require different hardware, different technique, different standard curve calculations that were fluorescent space. I said, "Norm, we don't need that. We don't want that. We want to be able to move either in our core technology or in our cartridge technology we want the customers to see a seamless transition from the natural to the synthetic." That's how Norm approached the project, and he was successful in doing that. In his creation of the Trillium, it would be a seamless transition for our customers as far as their hardware and software installation.
Mary:
That speaks to sustainability as well. But it was just like other people had created these artificial LAL tests, maybe only using one or two proteins. This is one where it uses all as far as we know, but it's one of those mysterious processes that, well, we know a lot about it. We don't perfectly understand how this cascade works. We just need to make sure that it works as well as the real thing. But anyway, Norm, keep going. I just want to give a little bit of background.
Norm Wainwright:
Yeah. Well, and just to build on your introduction, it was clear to me from the start that we had to really challenge ourselves and go after the entire cascade. For us, that meant there are three major enzymes in the cascade. This really gets to the beauty of why the horseshoe crab LAL natural system is so powerful. The cascade is important because as Foster said, the first enzyme the cascade interacts with endotoxin becomes activated to essentially activate at the second enzyme and the second activates the third enzyme. Each step, there's an amplification so that a tiny amount of endotoxin at the beginning can have that massive result of a blood clot that could be read in our system.
Again, I said the timing was important for this in two ways. One was that we really should be going to a full cascade. Secondly, the recombinant technology has progressed so far to be able to express foreign genes in systems to get them into production in large quantity, primarily driven by the biomedical industry. We had all those tools available to us and could basically read the sequence of the amino acids for each of those three key enzymes, create synthetic genes that could be inserted into a cell line that could then go into a fermentor in relatively large scale and produce consistent quality active enzymes that could be used to create a synthetic cascade of LAL. That's easy to say, but the challenge also is during that whole process, we have to be extremely careful that endotoxin doesn't seep into the system in some way, really in the same way that drug manufacturers have to be careful to keep endotoxin out of the process.
In our case, if endotoxin were introduced and a contamination occurred, that entire lot would basically be useless. We developed a lot of quality control tests that could be used along the way to ensure that the enzymes were active, they were not contaminated. We worked with some really good commercial partners to make that happen.
Mary:
In your development of the recombinant, were there any aha moments where it really started to come together? Any big discoveries that really helped the process along?
Norm Wainwright:
Well, yeah, I think the one that... I chuckle because there were quite a few, but I think the one that was really alien to us is as scientists working with LAL, we always had the natural material. It's a complete package to start with. When you have individual enzymes, the question comes, how do you know that those enzymes have the correct activity? How do you know what concentration to use? It's a big unknown at the present time in natural. We were at square one with very little information on how to proceed. We could make recombinant protein, but we then had to really assure ourselves that the activity was there and we had to mix it in the right proportion with the proper enzymes and pH and so on to actually make it function.
The first few stumbles out of the gate were a little bit worrying that, "Oh my gosh, what did we get into?" But I think the aha moment you mentioned was when we finally realized, "Okay, let's back up a second. Let's make sure we could analyze each of the enzymes separately and then begin mixing at different ratios and combinations to be successful." Finally, we started to have some success and to build on that, but it was quite a long process.
Mary:
To give a broader historical picture of how much research has been involved in this from the beginning. Before LAL became the standard, scientists were using the rabbit pyrogen test. Norm, can you describe the history of how we transitioned from rabbits to horseshoe crabs and how they compare? I know pyrogenic, for example, is different from endotoxin, so how did we make that leap?
Norm Wainwright:
Yeah, I'll certainly start with that, but I'm sure Foster can add some as well. But as Foster did already say, humans have a pyrogenic response to endotoxin, a fever that develops, and that's true of really all mammals. To test drugs originally for pyrogens, the drug manufacturers would inject into rabbits and measure their temperature. This was a relatively involved process. It used multiple animals for each test. It took quite a long time, several days, really, to get the results needed. It was expensive, not only in dollars, but in animals. When LAL was publicized, the discovery of Levin and Bang, again, this was a way that one could quantify endotoxin very quickly. Within an hour, a result could be obtained even in the early days, and it could be made quite quantitative. You get a number against a purified standard. It was, I guess, a big draw to, a pressure really, to move away from using so many animals with a less quantitative test, expensive and otherwise, and move to something that was invitro, fast, relatively inexpensive.
Knowing that, I guess there was some imperfection that is, many things can cause a pyrogenic response. But the most dangerous, I would say would be endotoxin. As Foster said, this can be a lethal situation if it were in high enough concentrations. The move to go toward LEL was a massive effort to prove to the scientific community that by measuring endotoxin, you could really predict the most dangerous pyrogenic responses that were being tested with the gold standard of the rabbit test. It took many years, many thousands of tests to do side by side showing that there was really an excellent correlation between measuring endotoxin with LAL and the pyrogen response that would be triggered if it were injected to animals.
Foster Jordan:
I think it was from what, the mid-seventies, enormous amount of comparative testing from the mid-seventies all the way through. The LAL test did not officially replace the rabbit pyrogen test until 1987. You're talking nearly over a decade worth of comparative testing before the FDA actually accepted as the official replacement to the rabbit, which was one of my biggest concerns as the business leader is as we transitioned from the natural LAL to the recombinant, is that we ensured that it was also not only correlated with LAL, we had to ensure that pyrogenicity was also equivalent because LAL has successfully replaced most all rabbit pyrogen tests. There is not a readily available way for us to go back and do what was done originally with natural LAL to the rabbit and do a comparative test.
It was critically important that we demonstrated, and I think that was a big challenge for Norm and his team, was to ensure that our Trillium reagent that they developed gave the similar results or gave the same results as our traditional reagents, because they've been out there for 40 years, they have a proven track record of being able to detect endotoxin. We've never had an instance where our reagents weren't able to detect contamination. We did enormous amount of comparative study. Norm, I think you had to tweak the formulas quite a few times to make sure that we were showing that our Trillium was actually giving the same results as our LAL.
Norm Wainwright:
No, that's absolutely true. All during the development process, as I mentioned earlier, we learned quite a bit in terms of how to mix the various enzymes together with the proper buffers and excipients that would allow that reagent, the mixture, to function well and to function efficiently. Ultimately, as Foster said, to compare it to pyrogenicity was really the final fine-tuning I would say that had to happen and will probably continue to happen throughout the future as this reagent becomes more and more refined.
Mary:
Yeah, so basically going from the gold standard of rabbits to long process to go to LAL and then into the recombinant, it just is having to go back basically to the beginning and start over again and make sure that it's just as safe as every other product that has been tested and is known.
Norm Wainwright:
Yeah, absolutely.
Foster Jordan:
Right. When we see differences, we really have to challenge those differences. Why are they there? Then the only way to really truly vet those differences is to go back to the original pyrogen test, and we do it very specifically and only when that necessary do we result to that, obviously, because we don't want to use rabbits unless they're necessary, but sometimes that's our only way of really challenging the differences.
Mary:
Yeah, unsurprisingly and justifiably the FDA and other regulatory agencies are very conservative when it comes to patient safety. They don't want to just replace something that they know works with something else unless they have very overwhelming evidence that it is just as good.
Foster Jordan:
Exactly. From our perspective, we're the market share leader in the LAL testing and endotoxin detection and is critically important to us that before we went to market with any product that we could ensure that it would perform as well as our existing product line.
Mary:
That leads into our next point of sustainability. That's, obviously, a concern whenever we are dealing with animal products. It's one of the Rs, really one of the three Rs. Foster, can you discuss how at each step in this historical process, sustainability has been improved, whether that was the goal or not?
Foster Jordan:
I think the original test when I joined the dominant test was the gel clot test. That test really is the test that requires the most raw material from the horseshoe crab because you need all the coagulogen, you need as much of the crude as you can to actually form that clot in the tube. If the clot, they used to call it the wet hand test, and at the end of the hour after you incubated your sample with LAL, you inverted the tube up 180 degrees and if the sample dumped into your hand, it was negative. If it formed a clot and stayed in the bottom of the tube, it was positive. Those reagents require enormous amount of crude. Over time, though, we were always conscious about optimization of the crude material, and we moved into turbidimetric reagents where you could get more tests per vial.
Then we moved to chromogenic reagents where you could cut the necessary crude by about half of what you needed for gel and tur because you were using a substrate that changed color instead of looking at turbidity or gel CLO formation. That allowed us to dramatically reduce the amount. Then as we moved into cartridge, that was a real ability to reduce by 95% because you use a combination of chromogenic reagents with microfluidics. We dramatically reduced the need for raw material.
If you look at the trajectory or the growth of the business, the growth of the business from a numbers of tests we produced annually to the numbers of crabs that we needed annually don't correlate because of our ability to actually get more out of the raw material year after year by evolving by the development of these new quantitative microfluidic techniques. Again, we expanded, we had one bleeding facility for many years, then two, but we ended up from one facility in 19 facilities around the world selling the reagents because we were able to drive the efficiency of the use of the raw material, which has always been a goal for us.
Mary:
Even the most material heavy of the original LAL tests used less material than the rabbit pyrogen. It was a constant stream of becoming more and more sustainable as time went on.
Foster Jordan:
Rabbits, just due to the cost of housing and just maintaining the colonies, the rabbit pyrogen test was really limited to finished product testing, Mary. You couldn't really use it as a high volume raw material screener because it was just too expensive. What the LAL did, too was improved the whole quality manufacturing process because you could test from every incoming raw material through all your in-process testing actually, as well as your finished product where you weren't able to actually do that without the pyrogen test.
Mary:
That's a good point. If you had a contamination earlier on the process, then you'd be wasting less material because you wouldn't bother carrying that batch through to the end of the process. Whereas you might end up throwing a lot of stuff away with the original version.
Foster Jordan:
Yeah, and think about it, your safety point was only at your final product at that point. You really had no quality data about pyrogenicity up through your manufacturing process. You really relied only on that finished product. With the LAL test, you really are pretty much guaranteed your finished product is going to be fine because you have all that in-process testing coming up. You rarely, as a result of endotoxin testing or LAL reagents now, you rarely ever see a finished product failure because there's so many in process control.
Mary:
Yeah. It also doesn't help that if you only test the finished product and there isn't contamination, you're not going to know where the contamination occurred. Whereas-
Foster Jordan:
That's true.
Mary:
If you tested each step, you might be able to figure it out. Anyway.
Foster Jordan:
That is true.
Mary:
A little bit of a tangent. I think it's important.
Foster Jordan:
Yeah, but that's true.
Mary:
Important to point out.
Foster Jordan:
Again, as far as corrective actions and deviations and manufacturing, the ability to have all those additional data points, to your point, is very important when you're trying to determine what the root cause of contamination is.
Mary:
All right, well if we can get into the realm of prediction or science fiction, what do you speculate could be the standard test for the future? How about you start, Norm?
Norm Wainwright:
Yeah. Well this would be a really tall order, for a number of reasons. First among them being that LAL has been so successful really over the past many decades on the safety front of protecting the drug supply and the medical devices that moving away from LAL would really require that we create something very much in order of magnitude better, for instance. From the beginning, again, if the rabbits served as a surrogate for establishing pyrogenicity for humans first and then LAL transition was really establishing an endotoxin test as a surrogate for the rabbit test results.
Now, we're basically fine-tuning the recombinant to transition where we can from natural to recombinant. The next transition, I would think, I'll just take a guess and say it would be interesting to have a more human specific test that if knowing that all of the information we have now from the human genome project, we have sequences for all of the genes, it would be really interesting to see if we could combine that with the knowledge of human immunology to choose a system that would mimic the cascade like effect of LAL, but rather with human constituents and be extremely specific again for the main purpose, which is establishing human patient safety.
Mary:
All right, any final thoughts, Foster, before we wrap up?
Foster Jordan:
Well, I think with our development of the cartridge and microfluidics and the regulatory situation between the natural LAL, I think we've given our clients a very good platform to be able to run a combination of the natural FDA licensed LAL for finished product release testing, as well as the ability to use the same system to test samples using our recombinant Trillium when they can, based upon where the regulations are at the time, whether it be raw materials or water. They can make that seamless transition over time to a completely recombinant solution.
That's going to take quite a while for, as you mentioned earlier, just the conservative nature of the regulatory environment of pharmaceutical, that transition's going to take a while, but to Norm's point, I think as our drug get more complex and cell and gene therapy and these drug treatments become more and more complex, will there need to be a better solution when it looks at the interaction between those complex drugs and person's immune system?
Is there a better solution to actually correlate it directly back to pyrogenicity and not just the looking for contamination with endotoxin? Because I think the natural reagents as well as the recombinant reagents will do a great job controlling the actual manufacturing process to ensure cleanliness. But the question is, as drugs get more complex and the interaction between people and these complex therapeutics, will we need some better test to look at pyrogenicity? The answer is probably yes. I think Norm's in the right direction on what we would actually need to develop to absolutely ensure as you're making these complex treatments that you don't have a pyrogenic reaction in patients.
Mary:
Yeah, good point. Good point. Well, thank you both so much for sharing your expertise with me. I really appreciate your time.
Foster Jordan:
Thank you, Mary. Thank you Norm.
Norm Wainwright:
My pleasure. If I could say one more word.
Mary:
Absolutely.
Norm Wainwright:
You've only really spoken with the two of us today, but for this project to have succeeded, it really touched on every single department within Charles River and dozens of people. The small numbers of research samples that we provided would not have made a product. For instance, John Dupecheck in manufacturing had a tremendous challenge to take those small numbers and make millions for the market and our QC department, customer support, shipping, you name it, everyone really pitched in to make this a reality, so kudos.
Foster Jordan:
Yeah. Agreed.
Mary:
Didn't you get real samples from clients donated basically to do the testing on, to test it against real world scenarios, basically?
Norm Wainwright:
Absolutely. Yeah.
Mary:
Yeah.
Norm Wainwright:
Customers made a huge impact in terms of the reality of real world samples and how they performed with recombinant and PTS.
Foster Jordan:
Yeah, patient safety was always the number one goal. We tested as many samples as we could possibly get our hands on.
Mary:
Excellent. All right, well thank you both so much again. It was a pleasure talking with you.
Foster Jordan:
Yep, thank you both.
Norm Wainwright:
Yeah, thank you.