Hayden Skalski- Life Sciences Product Application Specialist, Veolia Water Technologies & Solutions, Sievers Instruments
The link between horseshoe crabs and pharmaceutical and medical device manufacturing is an important subject today in the biomedical industry - an industry that is prioritizing sustainability while also ensuring safety and quality of products for patients. The blood from horseshoe crabs contains factors that are used to manufacture limulus amebocyte lysate (LAL), a reagent that can test whether drug products or medical devices have been contaminated with bacterial endotoxin. Through a series of cascade reactions, the crab’s blood factors have a clotting mechanism in place to detect any introduction of bacterial harm. This testing is critical to detect dangerous – and potentially deadly contaminants – but it puts pressure on horseshoe crabs and their desired blood.
The Atlantic horseshoe crab, Limulus polyphemus, is a species of marine arthropod found along the eastern coast of North and Central America. The Delaware Bay supports the largest spawning population in the world.1 The limulus species is around 450 million years old and is one of the most studied invertebrates in the world due to its unique blood. Horseshoe crabs are widely used for a few purposes such as a food source for birds (eggs), bait for commercial eel and conch fisheries, and the biomedical industry. Baiting of horseshoe crabs, particularly in the Northeast region, is allowed and is seen as the main cause of mortality and declining population of the Atlantic horseshoe crab species. Atlantic coastwide bait landings in 2020 were reported at 456,675 crabs.1 In the Southeast Atlantic state of South Carolina, harvesting of horseshoe crabs for any bait use is prohibited through law.2 All fisheries who capture and collect horseshoe crabs must possess a proper permit for commercial, educational, or private purposes.2 This law is extremely important to the horseshoe crab population, as it allows the population to flourish in the southeast and protects the crab species from baiting. As mentioned previously, a large population of the Atlantic horseshoe crabs is collected for the biomedical industry to support production of LAL reagent. Blood from the horseshoe crab is obtained by collecting adults and extracting a portion of their blood. Most crabs collected and bled by the biomedical industry are, as required by the Fishery Management Plant (FMP), released alive to the water from where they were collected; however, a portion of these crabs die from the procedure.1 Per the state compliance reports in 2021, the population of crabs have a very low mortality rate from biomedical use compared to the main source of mortality which is baiting. The 2019 Horseshoe Crab Benchmark Stock Assessment evaluated the stock status of the resource by region, finding populations within the Delaware Bay and Southeast regions remaining consistently neutral and good, respectively, through time.1
Due to the concerns of crab populations and the dependence on their blood for biomedical purposes, the awareness of alternative bacterial endotoxin methods has risen as of recent years. Recombinant Factor C (rFC) and recombinant LAL (rLAL) are two alternative methods which do not use horseshoe crab blood. rFC is the first clotting enzyme in the horseshoe crab’s blood clotting mechanism, and a clone of this Factor C is used for the rFC method. This alternative method is an end-point method, meaning one reading is taken at the beginning of the test, and then one at the very end. It works by a fluorescence reader amplifying a fluorogenic substrate when endotoxin is detected. Since this is an alternative method for detection of bacterial endotoxin, a company must go through additional and rigorous testing to show equivalency to the compendial BET methods. The FDA has recognized rFC as an alternative method, and states fi rms may use alternative methods and/or procedures if they provide advantages in terms of accuracy, sensitivity, precision, selectivity, or adaptability to automation or computerized data reduction, and in other special circumstances.3 If a company chooses to use the rFC assay, then alternative method validation should be performed in accordance with the requirements of USP <85>Bacterial Endotoxins Test and USP Chapter <1225>Validation of Compendial Procedures.3
Recombinant LAL reagent, or rLAL, is also an alternative test. It utilizes the same cascade as traditional LAL reagents. This means it contains all the factors that are responsible for detecting endotoxin that are found in horseshoe crab blood using a recombinant manufacturing process.4 This a step forward in the process of using a recombinant reagent as it contains all clotting factors and uses a traditional kinetic assay on an absorbance reader. Although it is a step forward in animal-free testing for BET, it is still considered an alternative method and companies must go through the same time consuming and rigorous process as rFC if they choose to use this assay. Since trying to validate and implement an alternative method for BET is not easy, some firms may ask what other solutions are currently available to help achieve a more sustainable and efficient way to test products for endotoxin.
The Sievers Eclipse Bacterial Endotoxins Testing (BET) Platform is one solution that offers significant advantages in terms of LAL reduction and compliance. The Eclipse reduces LAL use by up to 90 percent while remaining compliant with all global pharmaceutical regulations. This is a substantial improvement compared to traditional LAL methods such as the 96-well plate and gel clot. For the Sievers Eclipse BET platform, a novel microplate was designed that facilitates accurate and rapid dispersion of LAL and samples using centripetal force, metering chambers, and microfluidic channels. The microfluidic system enables users to carry out the same biochemistry that is performed in traditional endotoxin assays but with minimal hands-on effort, greater consistency, and drastically reduced LAL consumption. By decreasing horseshoe crab lysate use by up to 90%, the Eclipse platform reduces the demand on the most sensitive and unmatched natural endotoxin detection reagent on the planet. The Eclipse platform uses commercially available, FDA licensed LAL and meets all requirements of the harmonized global pharmacopoeia, USP <85>, EP 2.6.14 and JP 4.01. This empowers users to be conscious of today’s needs to protect valuable natural resources while still complying with the strict analytical and regulatory requirements drug and device manufacturers must meet.
References
- Atlantic States Marine Fisheries Commission. (n.d.). Horseshoe Crab. Retrieved from http://www.asmfc.org/species/horseshoe-crab
- Floyd, B., & DeLancey, L. (2012, March 1). South Carolina Horseshoe Crab Fishery and Management Program Compliance Report for the Year 2011. Retrieved from https:// www.dnr.sc.gov/marine/mrri/pubs/SCComplianceHSC2011.pdf
- U.S. Department of Health and Human Services Food and Drug Administration. (2012, June). Guidance for Industry: Pyrogen and Endotoxins Testing: Questions and Answers
- Associates Of Cape Cod Incorporated. (n.d.). Recombinant LAL Reagent PyroSmart NextGen™ Instructions For Use.
Hayden Skalski is the Life Sciences Product Application Specialist for the Sievers Instruments product line, specializing in bacterial endotoxins testing (BET). Hayden has over 8 years of experience in the pharmaceutical industry and Quality Control Microbiology and has presented on numerous topics surrounding endotoxin testing. Previously, Hayden held roles at Charles River Laboratories, Regeneron and Novartis, validating and executing method development protocols for endotoxin testing, providing customer support, troubleshooting and supporting high-volume product testing. Hayden has a B.S. from the University at Albany (SUNY) in Biology.
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