Most of today’s pharmaceutical and medical device manufacturers utilize limulus amebocyte lysate (LAL) – the blood from horseshoe crabs – to test for bacterial endotoxin. The testing is critical to detect dangerous and potentially deadly contaminants but, from a sustainability perspective, it puts pressure on the horseshoe crab population. The crabs are the most sensitive and unmatched natural endotoxin detection reagent on the planet. Most of those 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 procedure1. For this reason, alternative bacterial endotoxin methods have evolved in recent years. Recombinant Factor C (rFC) and recombinant cascade reagents (rCR) are two alternative methods that 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. It works by having a fluorescence reader amplify a fluorogenic substrate to detect the presence of endotoxin. The FDA has recognized rFC as an alternative method but does require equivalency testing to more common compendial BET methods, in accordance with the requirements of USP <85>Bacterial Endotoxins Test and USP Chapter <1225>Validation of Compendial Procedures2.
Recombinant cascade reagents, or rCR utilizes the same factors as traditional LAL reagents found in horseshoe crab blood, but by using a recombinant manufacturing process3, it is considered an alternative method. Using a recombinant reagent contains all clotting factors and uses a traditional kinetic assay on an absorbance reader, making it animal-free testing for BET. Companies that adopt this assay method also must go through the same validation/equivalency testing as rFC.
Although these methods are more sustainable for the horseshoe crab population, validating and implementing an alternative method for BET can be time consuming and rigorous, and leave many companies looking for a simpler, yet also sustainable option, for testing products for endotoxin.
The Sievers Eclipse Bacterial Endotoxins Testing (BET) Platform is a solution that offers significant advantages in terms of LAL reduction and compendial adherence. While not an alternative method, by performing a full IQ/OQ/PQ protocol and robust testing and validation similar to requirements of USP <1225>, users will have full confidence in its validation. The Eclipse uses commercially available, FDA licensed horseshoe crab LAL but reduces LAL use by up to 90 percent compared to traditional LAL methods such as the 96-well plate and gel clot. In developing the Eclipse, an ultramodern microplate was designed to facilitate accurate and rapid dispersion of LAL and samples using centripetal force, metering chambers, and microfluidic channels. The microfluidic system uses the same biochemistry as traditional endotoxin assays but with minimal hands-on effort, greater consistency, and drastically reduced LAL consumption– by up to 90% as mentioned.
The Sievers Eclipse Bacterial Endotoxins Testing (BET) Platform is fast becoming a BET testing method of choice because it allows pharmaceutical and medical device manufacturers to protect valuable natural resources and meet sustainability goals while complying with strict analytical and regulatory requirements of USP <85>, EP 2.6.14 and JP 4.01 harmonized global pharmacopeia.
Comparison of LAL Testing Methods
Author: Hayden Skalski- Life Sciences Product Application Specialist, Veolia Water Technologies & Solutions, Sievers Instruments
Hayden 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.
- References
-
1. Atlantic States Marine Fisheries Commission. (n.d.). Horseshoe Crab. Retrieved from http://www.asmfc.org/species/horseshoe-crab
2. U.S. Department of Health and Human Services Food and Drug Administration. (2012, June). Guidance for Industry: Pyrogen and Endotoxins Testing: Questions and Answers
3. Associates Of Cape Cod Incorporated. (n.d.). Recombinant LAL Reagent PyroSmart NextGen™ Instructions For Use.