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 with 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. Ultimately, health authorities linked a total of seven cases of illnesses to the pharmacy she had visited. 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.
Now, a platform made by Suez called the Sievers Eclipse Bacterial Endotoxins Testing Platform offers an easier way to conduct endotoxin testing at pharma facilities. Using groundbreaking technology that delivers elevated levels of automation and compliance, the Eclipse decreases the need for pipetting steps and is easy to use, reducing setup time by 85%. It also reduces the use of the required LAL reagent by 90%, which helps companies reach sustainability goals.
To learn more about the Eclipse, Pharma Manufacturing recently spoke to Hayden Skalski, the Life Science Product Applications Specialist for the Sievers line of Analytical Instruments at Suez.
Q: Why is the industry looking for improvements in endotoxin testing?
A: There are many reasons why improving the limulus amebocyte lysate (LAL) test is beneficial. Two main reasons are data integrity and more automation. The bacterial endotoxin test is a critical release test, meaning these life-changing medicines cannot be released to the public until they pass the LAL test. However, the traditional methods are extremely manual and require a significant amount of time to set up. Much of the analyst’s time in the labs will be spent setting up and running tests with these older techniques. And the more human intervention, the more likely the LAL test will be prone to errors.
And we have to remember that the LAL test is a very sensitive test — detecting down to one part per trillion. That’s equivalent to a grain of sand in an Olympic-sized pool, which means any small contamination introduced by the user will likely be detected
So by adopting newer technologies that are currently available today, such as the Eclipse microplate, QC labs can be assured that not only will they significantly reduce analysts’ hands-on time, but they will also reduce the percentage of potential errors, invalid tests and time spent retesting and writing investigations. This will ultimately free up lab analysts and allow them to focus on other specific tasks, such as SOP writing, change controls, etc.
Q: Let’s say a company decides to implement a new solution for LAL testing. What do you recommend they do in order to initiate that kind of change?
A: The easiest change would be going from the same method to the same method. An example is if you’re running a chromogenic 96-well plate and you want to switch to the chromogenic method, utilizing the Sievers Eclipse microplate, because you’re going from chromogenic to chromogenic and the test’s biochemistry is the same. It is then recommended to do a side-by-side study with your samples using both platforms to ensure the samples are suitable with the new solution. A change from gel-clot or turbidimetric to chromogenic requires a little more work, although it is still straight forward. Labs should run comparability studies to also ensure their products will be suited for a new platform/method.
Q: What are some of the important aspects of system validation in terms of LAL testing?
A: Bacterial endotoxin testing platforms must be validated to demonstrate that a valid endotoxin test can be carried out on the substances or products concerned. And you want a system that is fully compliant to ensure there is no doubt in its accuracy generating and maintaining 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.
Q: How does this platform fit into sustainability goals?
A: I like to start 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 cannot be sent to market until they’ve been tested for bacterial endotoxins. And horseshoe crab blood contains factors that, if extracted, could be used to manufacture what we call LAL — or limulus amebocyte lysate — which significantly improves our ability to test whether injectable drugs have been contaminated with endotoxin.
The vast majority of this reagent comes from the North American horseshoe crab, 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.
There are sustainability initiatives and even laws enacted to protect the crab so they can only be used for biomedical purposes, such as harvesting them for producing this important reagent. However, there is still a high probability that this species could become vulnerable to baiting and habitat loss. As such, it’s very critical that we find ways to conserve and optimize the use of this resource.
So instead of traditional LAL tests, other newer technologies, such as the Eclipse analyzer, offer LAL testing using up to 90% less LAL reagent. In addition, with just one vial of LAL reagent, a lab can run up to 63 samples on the Eclipse, compared to a 96-well plate that takes practically four vials for just for 21 samples. It’s vital that QC labs pursue newer, automated methods that use less reagent to help conserve this Limulus species for years to come.
Q: Can you tell me about some of the benefits of the platform’s microfluidic technology?
A: Microfluidics in endotoxin testing uses microfluidic liquid handling to facilitate accurate and rapid dispersion of the reagents and samples with drastically reduced volume to sample and LAL reagent within the Eclipse microplate. It’s achieved by using small microfluidic channels and metering chambers. Using centripetal force, the microplate spins really fast to control and automate all liquid measurements, flows, and mixing in preparation for analysis.
All standard curves and positive product controls, or PPCs, are embedded on the microplate for ease of use and the platform requires significantly less pipetting steps than traditional tests. In addition, the microfluidic liquid handling precisely measures all liquids for the end-user. That means that the precision typically required for physical action of pipetting is eliminated through the precise design of the Eclipse microfluidic microplate.
Q: Let’s wrap up by talking more about the Eclipse platform. How does it reduce the potential for human error and improve data integrity?
A: The Eclipse microplate comes preloaded with embedded standard curves ranging from 50 EU per ml down to 0.005 EU per ml from reference standard endotoxin, or RSE, and also contains positive product controls in duplicate up to 21 samples. So you don’t have to rely on an analyst creating a standard curve every assay. If the curve they generate is too potent or too weak, that can have a devastating effect on your test and may underestimate/overestimate the amount of endotoxin actually present. The Eclipse microplate takes care of that by having everything embedded, including the standard curve.
And by having that already set on the plate, you’ve removed the potential for a lot of human error. With traditional 96-well plate assays, every sample is spiked and duplicated manually. But we take that out of the picture too with embedded positive product controls, which leaves little room for error.
Also, with traditional plate assays, if you do a full plate of 21 samples, there are 242 pipetting steps for a full plate. With the Eclipse microplate, there are less than 30 pipetting steps, which reduces the risk for human error, the risk for repetitive injury and reduces the time spent executing the test.
By removing so many aspects of human intervention, the Eclipse drastically improves 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 to help adhere to those guidelines. When an assay is running, it transmits data to the software every five seconds, so you can check progress frequently throughout the assay to see its progress and see the data in real-time.
The software also has a routine database backup and restore function so data will not be lost. If a disaster occurred or something shut down the system, everything would be maintained.