[Solutions Spotlight] A groundbreaking approach to BET testing

Nov. 23, 2021

Endotoxin testing serves a vital function in pharma quality control by ensuring that products are safe for patients. But the traditional approach to Bacterial Endotoxins Testing (BET) is an area ripe for disruption and improvements. Now, the Sievers Eclipse BET platform by Suez offers new levels of automation, compliance and sustainability, ushering in the next generation of BET.


Meagan Parrish: If you want to understand why endotoxin testing in pharma is so important, a cluster of endotoxin poisoning cases that occurred in Australia provide a poignant example. In 2015, a 41-year-old patient who that day had received an infusion from a compounding pharmacy turned up in an ER room in Sydney. She had a fever, muscle twitches, vomiting, and pain in her abdomen, neck, and back. She was treated with broad-spectrum antimicrobial therapy and remained hospitalized for a week. Then, two weeks after being admitted, she developed a complication of the antibiotics C. diff, an excruciating inflammatory condition in the colon that can sometimes be fatal. Ultimately, health authorities linked a total of seven cases of illnesses to that pharmacy she had visited, and they noted that there were several lapses in aseptic production at the site and that a powder used to make the medication she was given contained high levels of endotoxins.

I'm Meagan Parrish, and you're listening to a special Solution Spotlight edition of Off Script, a Pharma Manufacturing podcast that goes beyond the pages of our magazine to discuss the issues that matter to the industry most. In this episode, we're taking a close look at a platform aimed at providing an easier way to conduct endotoxin testing and pharma facilities called the Sievers Eclipse Bacterial Endotoxins Testing Platform. The solution, which is made by Suez, includes a number of features that deliver both automation and compliance. Using groundbreaking technology, the Eclipse decreases the need for pipetting steps and is easy to install, reducing setup time by a whopping 85%. It also reduces the use of the required LAL reagent by 90%, which helps companies reach sustainability goals.

So, to learn more about the Eclipse, I'm joined today by Hayden Skalski, the Life Science Product Applications Specialist for the Sievers line of Analytical Instruments at Suez. Hayden has over eight years of experience in the pharma industry, and in quality control microbiology, and he's presented on numerous topics surrounding endotoxin testing.

Right, Hayden, thank you so much for joining me.

Hayden Skalski: Hey, Meagan, thanks for having me.

Meagan: Absolutely. Let's start by taking a look at the landscape for endotoxin tests. What are some of the options for endotoxin testing today and how do they compare?

Hayden: So currently, there are three types of approved endotoxin testing. One is the gel clot method and then two photometric methods. Gel clot was the first LAL test that was approved back in 1977. And to simplify that, how this method is performed, the LAL reagent is added to a test tube which contains a sample, and then that is incubated for an hour at 37 degrees. Upon that incubation completion, the tubes are inverted, and if the liquid has gelled or clotted, that would indicate the sample is contaminated with endotoxin. If no gel has formulated, then no endotoxin is present. This is a pass or fail test. So, you either get a positive or a negative test and that this is considered a qualitative test. So, it's either passing or failing.

The two photometric methods, which are more automated and give you a quantitative results so you get an actual numeric result, are the kinetic chromogenic method and the kinetic turbidimetric, which uses a spectrophotometer or an absorbance reader to perform the assay. The turbidimetric method measures an increase in turbidity or the time taken to reach a particular level of turbidity or onset time. The chromogenic method measures a color change, which would be yellow, over time. So, this kind of happens when a colorless chromogenic substrate is added to the LAL reagent that causes the yellow color to develop after a cleavage of the chromophore, which is known as parametric aniline or PNA. So, the more endotoxin that is present, the more yellow the solution will become. And generally, the turbidimetric and chromogenic techniques are run in the 96-well plate format and they are very time-consuming tasks.

There are newer technologies that have been developed to run these LAL tests and those are a cartridge-based technology and a microfluidic plate, such as the Sievers Eclipse. There are also some recombinant assays out there, which use a synthetic form of factor C instead of actual blood cells that is found in the horseshoe crabs' amebocytes that is responsible for the enzymatic reactions to detect endotoxin. And this type of technology uses a fluorescence reader, which is separate from the absorbance readers used in traditional LAL methods. This method, however, is not compendial and it requires to undergo an alternative method validation per USP 1223.

Meagan: Yeah, and I was just about to ask you about some of the challenges and limitations associated with these different methods. Can you talk a little bit about that and tell me why the industry is looking for improvements on endotoxin testing?

Hayden: Yeah, so there are many reasons why improving the LAL test is so beneficial and why the industry is striving to improve it. So, the bacterial endotoxin test is a critical release test, meaning these life-changing medicines and these products cannot be released to the public until it passes the LAL test. So, however, with the traditional methods, like I described earlier, such as gel clot and the 96-well plate methods come from caveats. And these other techniques are extremely manual and they require a significant amount of time to set up these tests. So, a lot of the analysts in the labs will spend most of their day kind of setting up and running these older techniques. So I have to say, you know, the more human intervention, the more likely the LAL test is prone to errors. And we have to remember that the LAL test is a very sensitive test, detecting down to one part per trillion, so that's equivalent to a grain of sand in an Olympic-sized pool. That is pretty remarkable if you think about it. However, that also means any type of small contamination by the user will most likely be detected. So companies, you know, want to get their product out to market faster to help these patients, but if there are failed or invalid LAL tests, this means a lot of retesting, a lot of investigations, which means the product is getting held up.

So by adopting newer technologies that are currently available today, such as the Eclipse microplate, the QC labs can be assured that not only will it significantly reduce analysts' hands-on time, but it will also reduce the percentage of potential invalid tests and time spent retesting and writing all these investigations. Now, the time not spent doing those two tasks will also free up analysts to run more LAL tests and focus on other lab-specific tasks.

And I like to just talk about kind of another reason why this industry seeks to improve LAL testing and that is data integrity. How this can be tied back into human error and that a lot of these processes tied into LAL testing allow for this error to occur. So, as labs use more outdated methods, such as gel clot, they are subjecting themselves to these data integrity issues and a lot of human error due to the manual processes of the test. The gel clot test, like I just mentioned, it's subjective meaning it's based on analyst observations. And this observation needs to be verified by second analysts, which they may interpret the results differently. And, you know, as kinetic methods are introduced, and as newer technologies are introduced, the subjective aspect goes away. And the new data software that is tied into these newer methods that interpolate the data, which ties back to the software and is manually read by the analysts, and this ties into the 21 CFR Part 11. So, maintaining that data integrity compliance.

Meagan: Yeah, so lots of ways that the method can be improved upon and lots of ways this technology really helps. So, let's say that a company decides that they're going to go ahead and implement a new solution for LAL testing or switch from one solution to another. What do you recommend that they do in order to go ahead and initiate that kind of change?

Hayden: Yeah, so I can start off with the easiest change that a lab would be looking to do, and that would be going from the same method to the same method. So, example here is if you're running a chromogenic 96-well plate and you want to switch to the chromogenic method, utilizing the Siever Eclipse microplate, this is the easiest because you're going from chromogenic to chromogenic. And that pretty much means the biochemistry is still the same and still chromogenic, so that is going to be the easiest switch, and that is recommended to do that as a side-by-side study of the current platform you're using with your samples and using the new validated platform that you want to switch to to ensure the product is suitable. And since you are using the same biochemistry in the chromogenic method, a one lot validation would be sufficient at the same dilution if the results are comparable.

Another example is if you are currently testing a gel clot method or kinetic turbidimetric and you're looking to switch to kinetic chromogenic using the Eclipse platform. This requires a little more of a comprehensive approach, but it's still easily attainable. So, what you want to do in this situation is a method suitability test. And this basically means you're going to perform this suitability test, which is also known as an inhibition enhancing screen at different dilutions to show that the products can be accurately tested and recover endotoxin using the new method and that there is no interference. So, once a suitable dilution is chosen using the new technique, you will perform a three-lot revalidation of the product.

I like to say it all doesn't have to get done at once, you know, labs can pick and choose at their convenience when they do this revalidation to make it more suitable and time worthy for them. And when you're changing methods, or you're changing lysate vendors or you're switching platforms for a drug product, QC labs often think it's a daunting task and a complicated process due to the regulatory filings. So, it is a recommendation when including your product in a filing for endotoxin to state briefly how it is tested. So, an example is you would say drug XYZ utilizes one of the compendial photometric kinetic methods for LAL testing. That way if you're ever switching vendors or methods, it makes it easier to change and you're not tied into one specific vendor or specific method. Now there is some guidance on how to go about changing a filing and it is a guidance for the industry by the FDA called changes to an improved NDA or ANDA.

There are two types of filing changes and that is a moderate change and a minor change. So, a change from the gel clot method to a turbidimetric or chromogenic method would fall under a moderate change known as CBE 30 or CBE zero and the CBE stands for changes being effective. The CBE 30 requires submission of a supplement to the FDA at least 30 days before the distribution of the drug product made using the change. A CBE zero would be considered as going from a 96-well plate chromogenic assay to the Eclipse chromogenic assay product. Now for the CBE zero, the FDA may identify certain moderate changes for which distribution can occur when the FDA receives its supplement. So in other words, the FDA will approve the LAL changes as long as the new method is compliant with USP 85. Now, an annual report is the last filing change and it's considered a minor change, a company will submit an annual report to the FDA telling them of the changes that were made. So switching lysate vendors would fall under this category, and going from a 96-well plate KCA to the Eclipse KCA product can also be considered in an annual report as well. So a lot of companies and their quality assurance or the regulatory affairs department will have their own internal requirements, we're going about how to do a filing change and their filing change could be a CBE 30 for LAL vendor change compared to doing an annual report. So, it all depends on the internal specifications for your company.

Meagan: Okay, great. And I was going to ask you about system validation. I mean, you touched on this a little bit, but I wondered if you wanted to add anything. So, tell me a little bit about some of the important aspects of system validation in terms of LAL testing.

Hayden: Bacterial endotoxin testing platforms so that they must be validated to, you know, demonstrate that a valid endotoxin test can be carried out on the substances or products concerned. Now, this may entail a procedure for removing interference, and you want a system that is fully compliant with 21 CFR Part 11 and Data Integrity guidelines that align with ALCOA Plus to ensure there is no doubt with regards to accurately generating and maintaining that data. You also want to ensure that the system can demonstrate linearity, accuracy, precision, sensitivity, equivalency, and a one-to-one lysate to sample ratio. Now, this will all fall under the IQ, OQ, PQ system validation. And this is extremely important, you know, to successfully carry out these validation requirements.

Meagan: Great. And of course, sustainability is a huge topic right now. In fact, I mean, sustainability used to be one of those things that businesses worked into their strategy if they could, but now it's kind of a given that companies have produced robust sustainability programs. So, tell me how this fits into sustainability goals. And what do you think is important in regards to endotoxin testing and sustainability?

Hayden: Of course. I like to just start, you know, by saying, you can thank a horseshoe crab, if you've ever received the recent COVID vaccine or IV treatment, or any injectable drug or medical device. These products, they cannot be sent out to market until they've been tested for bacterial endotoxins, which if they were sent out in the market and they were contaminated, it would cause a fever, adverse reactions and even death in a patient. So, very important for these products to be tested for endotoxins. Now, the horseshoe crab blood contains factors that if extracted could be used to manufacture, what we call LAL, so that stands for Limulus amebocyte lysate, and that's an aqueous in-vitro reagent that holds the potential to significantly improve our ability to detect whether injectable drugs have been contaminated with endotoxin. Now, the vast majority of this reagent comes from the North American horseshoe crab, Limulus Polyphemus, which is found up and down the eastern seaboard of the United States. So, as vaccines ramp up worldwide and new treatments for diseases emerge, there's an increasing demand for the horseshoe crabs' unique blood.

Now, there are sustainability initiatives and even laws enacted to protect the crab so they cannot be used for bait or fishery practices and they're in place so they can only be used for biomedical purposes, such as harvesting them for producing this important reagent. However, there is a still high probability that these species becomes vulnerable to baiting and habitat loss, which could lead to a decline in population. As such, it's very critical that we find ways to conserve and optimize the use of this resource as much as possible. So, instead of traditional LAL tests, which is gel clot and 96-well plate assays I touched on earlier, other newer technologies, such as the Eclipse analyzer, that now offer LAL testing using up to 90% less LAL reagent than those traditional methods. It's vital that QC labs pursue newer automated methods that use less reagent. Microfluidic technology that utilizes small volumes of samples and reagents in small spaces will help conserve this Limulus species for years to come, as well as continued monitoring of the horseshoe crab to protect it. So, life-saving medicine does depend on this crab and the reagent that it produces, and industry as a whole, we must work together on conserving this special creature by looking to newer technologies that utilize less reagent.

Meagan: Now, I'm going to circle you back to the topic of validation. I was wondering if you could tell me why is it important to perform product validation?

Hayden: Sure. So, product validation, this is also known as, you know, product screening or some of us like to call it the inhibition enhancement screening. And this is performed on new products or existing products that are going to be changing LAL methods. So this typically involves three lots of products and these are screened at different dilutions to see which dilution is not interfering and gives the best spike recovery. And the best spike recovery is ideal to be around 100%. So, the product validation pretty much or basically gives validity to the test ensuring the product is suitable at that dilution, and it can recover endotoxin accurately. Now, most products, aside from water will interfere with the LAL test. So, it's very important to do this type of screening. Unfortunately, the LAL assay is very sensitive, and most often than not, if you do have interference, it can be overcome with simple dilution. So the inhibition enhancer screening is just that, you know, you're screening the product to see if it interferes with the LAL reagent that will inhibit endotoxin recovery less than 50% because in LAL tests, we have a range of 50% to 200%, we want to be inside that range. So, if you're less than 50%, that's going to inhibit the LAL reagent that's interacting with your products. And we also want to see if it's going to enhance the recovery, so greater than 200%. Now, the main concern with inhibition is it could underestimate the value of endotoxin and the product could potentially be released to the market. So, we call it a false negative. So, if there's something, you know, inhibiting that reaction, it might give you a passing test, which in real-time, it's interfering, so we call that a false negative and that could go out to the market and be released and that will be a big problem. And now enhancement is much less dangerous inhibition because as a product will not be released to the market, it'll just show, you know, levels of recovered endotoxin. And nevertheless, you know, the screening is done during the validation stage and must pass all criteria outlined in chapter 85 of the USP before being validated. So you'll get to see during that screening if something's being inhibited or enhanced.

Meagan: Okay, great. And I want to talk a little bit more about the technology that is behind this platform, the microfluidic technology. Can you tell me what some of the benefits are of that and also just a bit more about how it works?

Hayden: Sure. So, microfluidics in endotoxin testing is microfluidic liquid handling that facilitates accurate and rapid dispersion of the reagents and samples with drastically reduced volume to sample and LAL reagent within the Eclipse microplate. So, it's achieved by using all these little microfluidic channels and metering chambers, and they use the centrifugal force. So, the microsuede spins really fast to control and automate all liquid measurements, flows, and mixing in preparation for analysis. And microfluidic technologies automation in the simplest way. So it simplifies endotoxin testing in a platform that is extremely easy to set up and use without the need for robotics. All standard curves and positive product controls or PPCs are embedded on the microplate for ease of use and comes with significantly less pipetting steps than traditional tests. In addition, you know, the microfluidic liquid handling precisely measures all liquids for the end-user. And that means basically, that the precision typically required for physical action of pipetting is eliminated through the precise design of the Eclipse microfluidic microplate.

Meagan: Okay, great. And at the beginning of this conversation, we talked a bit about the benefits of platform technologies like Eclipse and some of the ways in which they're improving endotoxin testing. So, I want to wrap up by asking you to elaborate on that a little bit more. And in particular, I was wondering if you can tell me how the Eclipse reduces the potential for human error and also improves data integrity?

Hayden: Absolutely. The Eclipse microplate comes preloaded with embedded standard curves ranging from 50 EU per mil down to 0.005 EU per mil from reference standard endotoxin, or RSE, and also contains positive product controls and duplicate up to 21 samples. So, you don't have to rely on an analyst creating a standard curve every assay. You know, by doing that, you know, you can have consequences if the curve they generate is too potent or too weak, and that can have a devastating effect on your test. So, the Eclipse microplate takes care of that for you by having everything embedded. So, that basically, by having that already set on the plate, you're already removing potential for, you know, a lot of human error. Now, with traditional 96-well plate assays, every sample is spiked and duplicated manually. So, any small distraction, you know, could lead to a miss-spike well, and that leads to an invalid sample and then investigation. So, we take that out of the picture, too. So, that really leaves little room for error. Now also, with traditional plate assays, if you do a full plate of 21 samples, that is a total of 242 pipetting steps for a full plate. With the Eclipse microplate, there are 27 pipetting steps. So, this reduction in pipetting steps not only reduces the risk for human error enormously but also reduces the risk for repetitive injury and it reduces a lot of time spent executing the test.

But kind of just to wrap that part up, you know, by removing so many aspects of human intervention, the Eclipse drastically improves, you know, data integrity on that end. And just speaking, you know, a little more on data integrity, the Eclipse platform comes with the Eclipse software to analyze endotoxin, and the Eclipse software offers a full 21 CFR Part 11 data integrity compliant package that includes ALCOA Plus principles and adheres to those guidelines. So, you know, when an assay is running, it transmits data to the software every five seconds. So, you can go check progress frequently throughout the assay to see how it's doing and see the data in real-time. The software also has a highly customizable permission and product accessories database, in which a lab supervisor can set up different analysts to test only certain products or samples and give different permissions. So, it's really up to the lab, how they want to set it up. But at least, you know, we give them all types of accesses, and they get to pick and choose how they want to run and set that up. And just to kind of conclude it, the software, you know, has routine database backup and restore so data will not be lost, if there was a disaster to occur or something shut down the system, everything will be maintained. So it really ties back into, to very data integrity compliant overall.

Meagan: Well, that's great, a lot of great improvements with the Eclipse software it sounds like, or the Eclipse platform rather. Thank you so much for taking the time today to explain why this is such an important technology and why the issue of endotoxin testing is so critical for the pharma industry.

Hayden: Yeah, thank you so much for having me on, and I was happy and excited to discuss all these important questions surrounding endotoxin testing.

Meagan: Yeah, of course.

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