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article imageQ&A: Importance of endotoxin testing COVID-19 vaccines Special

By Tim Sandle     Aug 26, 2020 in Science
Bacterial endotoxin presents a risk to sterile medicine products, especially those administered intravenously or intrathecally. Assessing endotoxin levels is also important in establishing if new COVID-19 vaccines are safe.
Endotoxin refers to a component of the outer cell membrane of Gram-negative bacteria called lipopolysaccharide (LPS). LPS is the biologically active portion of naturally occurring endotoxin complexes. One portion of liposaccharide called Lipid A exerts a powerful biological response modifier that can stimulate the mammalian immune system, triggering fever or endotoxic shock. The presence of endotoxin in sterile pharmaceutical products presents a significant risk to patients, and this includes vaccines. During preclinical development of the next generation vaccines endotoxin levels should be closely monitored. Hence, consideration of endotoxin contamination represents an important critical quality attribute as companies attempt to produce a vaccine against SARS-CoV-2.
To look into the issue further, Digital Journal spoke with Nicola Reid, Associate Director Product Management, Endotoxin Products, Microbial Solutions at Charles River, about their work in Charleston, SC, U.S.
Digital Journal: What is endotoxin and why is the detection of lipopolysaccharide important in relation to vaccine development?
Nicola Reid: Endotoxin is derived from the outer membrane of Gram-negative bacteria. It is a lipopolysaccharide (LPS) due to the fact that it is made up of a lipid and two polysaccharide entities. The structure of endotoxin varies among each species of bacteria. Endotoxin is a pyrogen--the word pyrogen comes from the Greek word for burning or fire, as it is defined as any substance that causes an increase in body temperature or produces a fever. It is important to test for endotoxins in vaccine development because if levels of endotoxin are over a specified level in the product, this could cause fever along with other immune responses in patients who receive the vaccine.
Horseshoe crabs mating in the Delaware Bay of Southern New Jersey.
Horseshoe crabs mating in the Delaware Bay of Southern New Jersey.
Asturnut (CC BY-SA 3.0)
Digital Journal note: Endotoxin levels above a certain threshold can also cause endotoxin shock; research suggests that half of the cases of septic shock are caused by bacteria that produce LPS, also known as endotoxin.
DJ: How do the amoebocytes of the horseshoe crab detect endotoxin?
Reid: Horseshoe crab blood is complex and contains four primary factors. It has factor C, factor B (amplification - binds to the surface of LPS), pro-clotting enzymes, and a coagulate that actually forms the clot at the very end. In addition, there are many intercellular Inhibitors (LICI) that contribute to the intermolecular autocatalytic activation that causes a clot. It’s a sophisticated biological model that allows us to effectively test a broad range of endotoxins. 440 million years of evolution have given us this unique and multifaceted system, and each component has a purpose in the detection of all endotoxins.
Digital Journal note: There are four species of horseshoe crab in existence: Carcinoscorpius rotundicauda (South and Southeast Asia); Limulus polyphemus (U.S. Atlantic coast and in the Gulf of Mexico); Tachypleus gigas (South and Southeast Asia); and Tachypleus tridentatus (China and Japan). Most diagnostic reagents are produced from Limulus.
To test for endotoxin levels, Charles River produce instrumentation and test kits, with one of the devices called the PTS, a point-of-use spectrophotometer.
Endotoxin test device (PTS) in Tim Sandle s laboratory.
Endotoxin test device (PTS) in Tim Sandle's laboratory.
DJ: How does the Endosafe PTS work?
Reid: All of our Endosafe® Cartridge technology instruments (nexgen-PTS™, nexgen-MCS™ and Nexus™) are incubating spectrophotometers that utilizes LAL kinetic chromogenic methodology to measure colour intensity directly related to the endotoxin concentration in a sample. Each cartridge is pre-loaded with all of the reagents required to perform a quantitative LAL test in 15-minutes, eliminating preparation of multiple reagents and reducing the opportunity for technician error.
Digital Journal note: LAL is a reference to the test method and the reagent to assess endotoxin, with LAL being an initialism for Limulus amoebocyte lysate. This active ingredient is produced biochemically, and commercial LAL differs according to each manufacturer.
DJ: How does the PTS help with Limulus crab conservation?
Reid: The cartridges use 95 percent less LAL than a traditional LAL Assay. By reducing the amount of LAL used for a test, it reduces the amount of bleeding of the Atlantic Horseshoe crabs that needs to take place. Any global increases in testing can easily be absorbed when only 5 percent of the lysate is needed, compared to traditional LAL tests. If all tests were performed using this optimized technology, today’s entire worldwide demand could be met with the blood from less than our current annual quota.
Digital Journal note: Approximately 500,000 Limulus are harvested annually for manufacturing the LAL reagent. Estimates about mortality rates vary.
DJ: Can the PTS be used for large scale testing?
Reid: Our suite of Endosafe Cartridge technology instrumentation range from single sample testing with the nexgen-PTS, to 5 sample testing simultaneously with the nexgen-MCS to a fully automated Nexus system capable of testing 60 samples in 3 hours.
DJ: How do your reagents and technology differ from your competitors?
Reid: We truly believe we have a responsibility to preserve, protect, and live harmoniously with the animals that share our planet. Charles River is proud to play an instrumental role in alleviating pressures on horseshoe crab populations through expanded conservation and educational efforts, active animal welfare campaigns and decades of research and development. With the introduction of microfluidic testing through cartridge technology which is unique to Charles River. We will continue to investigate ways to reduce the amount of LAL required for bacterial endotoxin testing. We carry out large amounts of R&D work to ensure robustness and repeatability with our products and provide the highest level of customer support.
DJ: Is recombinant lysate the future of endotoxin testing?
Reid: With how technology is advancing, Recombinant lysate can be the future of bacterial endotoxin testing, however before we can proceed with that we must ensure that the recombinant reagents available are equivalent to LAL. Currently studies only show suitability but not comparability to LAL for endotoxin testing. We must continue to develop these reagents and prove equivalency over many test points to ensure the safety of patients before the move is made. We must also ensure the quality of these new reagents, LAL is currently licensed by the FDA but recombinant reagents will not have this same licensing and therefore the responsibility to prove quality is on the end user. I believe this can be achieved but we are not there yet.
Digital Journal note: A synthetic version of LAL based on a recombinant form of the major endotoxin-reactive component of C. rotundicauda has been developed and is commercially available. Some laboratories use recombinant lysate, others use lysate derived from horseshoe crabs.
More about endotoxin, Bacteria, Covid19, Vaccine
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