Q&A covering past, present, and future perspectives on the LAL reagent
From American Pharmaceutical Review:
Since the introduction of the LAL reagent roughly 50 years ago, three methods of Testing have been primarily used. how has your company used these methods?
JD: Actually, we use all four methods: gel-clot, kinetic turbidimetric, kineticchromogenic – and the PTS™. These methods are used to test and release incoming raw materials and to evaluate intermediate formulation buffers that are subsequently used to produce licensed LAL products. Our accessory products also benefit from the use of these methods prior to release. Finally, we leverage all of these techniques in developing and validating LAL test methods for customer-specific products.
TS: We began with the gel-clot test, which worked fine for water samples although it proved to be somewhat laborious for testing final products. Bio Products Laboratory was one of the first laboratories in the UK to adopt turbidimetric testing. This was in 1990, and technologies have improved greatly since then. We use turbidimetric testing today to test water samples (WFI and purified), intermediate products, finished product, and for other applications, such as testing container-closures for levels of endotoxin. We have also used LAL to screen for glucans.
How did the introduction of the LAL test change our industry?
TS: In terms of moving away from rabbit pyrogen testing this was very significant. It not only resulted in less animal testing being conducted, it also sped up the time-to-result and placed control of the test back into the hands of individual companies. Moreover, LAL results – where endotoxin is the pyrogen of concern – are more accurate and can be trended.JD: The LAL test has dramatically improved the quality of drug products and medical devices since its introduction. It has allowed the production process to be monitored in a way not possible before, resulting in better and safer products entering the healthcare system.
Furthermore, the relative low cost of the test allowed more samples to be tested. This expanded the scope of in-process control testing.
What are the key trends in endotoxin and LAL testing? What advances have been made?
JD: A major trend in LAL testing today is the continual movement away from the traditional gel-clot reagents and into more rapid quantitative methods. The data from these systems is also becoming more and more directly interfaced with a company’s LIMS system for more sophisticated data management and electronic record keeping. Obviously, it’s difficult to top the invention of LAL and replacement of the rabbit pyrogen test; however, there have been two significant LAL developments that really stand out. One is the development of quantitative LAL-based methods, which allow customers to quantify levels of endotoxin in their samples below their limits and to look for trends that could proactively prevent failures. Following that, the FDA approval of the Endosafe®-PTS™, the first point-of-use endotoxin detection system, allows users to rapidly test for the presence of endotoxin in the manufacturing environment for real-time results. Celebrating its 10-year anniversary, the Endosafe®- PTS™, has revolutionized the healthcare industry, enabling QC laboratories to improve sample management, decrease testing time and accelerate product production while still being compliant with global pharmacopoeial methods.
TS: In terms of trends, the scope of testing seems to be everincreasing with more tests being conducted.
Regarding advances, in recent years these have been towards rapid methods and with test systems having a larger capacity, to ease sample throughput. Recombinant lysates have also appeared on the market which seems as effective as lysate derived from the Limulus crab.
LW: Robust and more sensitive assays are the top two trends in the endotoxin detection industry; while portable systems and comprehensive analytical tools, like WinKQCL™ Endotoxin Detection and Analysis Software, are also becoming more essential to end-users.
In addition, there is a growing awareness of the need to conserve diminishing animal sources that are used for pharmaceutical and medical device testing. Bacterial endotoxin testing is rapidly evolving from an industry that relied heavily upon animal resources, i.e. rabbits and horseshoe crabs, to the development of biochemical assays as an endotoxin detector. This is evident in the increasing number of endusers who are now evaluating and validating alternative methods, like recombinant Factor C and the Monocyte Activation Test (MAT), instead of LAL-based tests for endotoxin detection. A little over ten years ago, Lonza recognized this progression of the industry and was the first company to develop a recombinant method for endotoxin detection that did not rely on the blood from the horseshoe crab: the PyroGene™ Recombinant Factor C Assay. Factor C, the first component of the horseshoe crab clotting cascade activated by endotoxin, is the critical component that allows for detection of endotoxin without isolation of LAL from the horseshoe crab. Endotoxin detection is more specific with the recombinant Factor C assay because there is less interference from other substances, like beta (β)-glucans. In addition, recombinant Factor C assays are less variable, as compared to the LAL test which may perform differently based on pooling of various lysates, while still providing the sensitivity end-users require.
JD: Actually, we use all four methods: gel-clot, kinetic turbidimetric, kineticchromogenic – and the PTS™. These methods are used to test and release incoming raw materials and to evaluate intermediate formulation buffers that are subsequently used to produce licensed LAL products. Our accessory products also benefit from the use of these methods prior to release. Finally, we leverage all of these techniques in developing and validating LAL test methods for customer-specific products.
TS: We began with the gel-clot test, which worked fine for water samples although it proved to be somewhat laborious for testing final products. Bio Products Laboratory was one of the first laboratories in the UK to adopt turbidimetric testing. This was in 1990, and technologies have improved greatly since then. We use turbidimetric testing today to test water samples (WFI and purified), intermediate products, finished product, and for other applications, such as testing container-closures for levels of endotoxin. We have also used LAL to screen for glucans.
How did the introduction of the LAL test change our industry?
TS: In terms of moving away from rabbit pyrogen testing this was very significant. It not only resulted in less animal testing being conducted, it also sped up the time-to-result and placed control of the test back into the hands of individual companies. Moreover, LAL results – where endotoxin is the pyrogen of concern – are more accurate and can be trended.JD: The LAL test has dramatically improved the quality of drug products and medical devices since its introduction. It has allowed the production process to be monitored in a way not possible before, resulting in better and safer products entering the healthcare system.
Furthermore, the relative low cost of the test allowed more samples to be tested. This expanded the scope of in-process control testing.
What are the key trends in endotoxin and LAL testing? What advances have been made?
JD: A major trend in LAL testing today is the continual movement away from the traditional gel-clot reagents and into more rapid quantitative methods. The data from these systems is also becoming more and more directly interfaced with a company’s LIMS system for more sophisticated data management and electronic record keeping. Obviously, it’s difficult to top the invention of LAL and replacement of the rabbit pyrogen test; however, there have been two significant LAL developments that really stand out. One is the development of quantitative LAL-based methods, which allow customers to quantify levels of endotoxin in their samples below their limits and to look for trends that could proactively prevent failures. Following that, the FDA approval of the Endosafe®-PTS™, the first point-of-use endotoxin detection system, allows users to rapidly test for the presence of endotoxin in the manufacturing environment for real-time results. Celebrating its 10-year anniversary, the Endosafe®- PTS™, has revolutionized the healthcare industry, enabling QC laboratories to improve sample management, decrease testing time and accelerate product production while still being compliant with global pharmacopoeial methods.
TS: In terms of trends, the scope of testing seems to be everincreasing with more tests being conducted.
Regarding advances, in recent years these have been towards rapid methods and with test systems having a larger capacity, to ease sample throughput. Recombinant lysates have also appeared on the market which seems as effective as lysate derived from the Limulus crab.
LW: Robust and more sensitive assays are the top two trends in the endotoxin detection industry; while portable systems and comprehensive analytical tools, like WinKQCL™ Endotoxin Detection and Analysis Software, are also becoming more essential to end-users.
In addition, there is a growing awareness of the need to conserve diminishing animal sources that are used for pharmaceutical and medical device testing. Bacterial endotoxin testing is rapidly evolving from an industry that relied heavily upon animal resources, i.e. rabbits and horseshoe crabs, to the development of biochemical assays as an endotoxin detector. This is evident in the increasing number of endusers who are now evaluating and validating alternative methods, like recombinant Factor C and the Monocyte Activation Test (MAT), instead of LAL-based tests for endotoxin detection. A little over ten years ago, Lonza recognized this progression of the industry and was the first company to develop a recombinant method for endotoxin detection that did not rely on the blood from the horseshoe crab: the PyroGene™ Recombinant Factor C Assay. Factor C, the first component of the horseshoe crab clotting cascade activated by endotoxin, is the critical component that allows for detection of endotoxin without isolation of LAL from the horseshoe crab. Endotoxin detection is more specific with the recombinant Factor C assay because there is less interference from other substances, like beta (β)-glucans. In addition, recombinant Factor C assays are less variable, as compared to the LAL test which may perform differently based on pooling of various lysates, while still providing the sensitivity end-users require.
What new challenges will be faced in the years to come?
JD: The development of new pharmaceutical drug products is becoming more and more sophisticated. These newer products, including biologics providing highly targeted therapies, involve spectacularly expensive production processes. Our challenge is to provide customers with all of the tools necessary to detect and, thereby, mitigate gram-negative bacterial contamination. These efforts require rapid methods, robust LAL testing methodologies, and the means to track and trend in-process and final-product endotoxin testing. Regulatory bodies currently emphasize process understanding, capability and control. These crucial production characteristics will become even more critical in the years to come.
TS: I think that recombinant lysates will become more commonplace, given some concerns about horseshow crab ecology. I also think that innovations will be made with rapid methods, and applied applications like on-line testing of water systems with fixed endotoxin reading devices. It is possible that robotic LAL testing – a fully automated method – will emerge (this was discussed a few years ago but it didn’t really develop). It is possible that the Monocyte Activation Test will challenge LAL as a key test for pyrogens, although adoption of this method has been slow and there are some concerns about its variability. Whatever happens in the future, the need to carry out endotoxin testing will remain as a key release test for many pharmaceutical products.
LW: New pharmaceutical and medical devices are continually being developed and released into commerce after regulatory approval. While some are approved, many more go through the same development process and clinical trials and are not approved. However, all of these products require BET testing if they are parenterally administered or implantable medical devices. Given the recent and expected future increased demand for LAL, the need to develop a sustainable alternative to the LAL method is a growing concern for end-users and regulators.
The distribution of horseshoe crabs is not worldwide. Since the population is limited by a lack of geographical distribution as well as the size of the population, any environmental threat provides a risk to the supply of available HSC. Given the demands being put on the limited HSC resource, as well as the risks to that resource, other options must be explored to meet the demands of the Pharmaceutical and Medical Device industries, and ultimately of the patients that rely on a safe supply of those products.Posted by Dr. Tim SandleContinued growth in the number of tests performed annually and increasing restrictions on fishing quotas for horseshoe crabs are mutually incompatible. The Asian species of horseshoe crabs, TAL, are now reported to be depleted, while the population of LAL on the East Coast of the US also had a significant decline in the 1980s and 1990s.
