Testing the Tests for Contaminated Water
Do synthetic bacterial endotoxin tests match the accuracy of the gold standard LAL?
The current standard for endotoxin testing is the limulus amebocyte lysate (LAL) test, derived from the natural ability of horseshoe crab blood to clot in the presence of endotoxins. LAL tests were developed in the 1970s as an alternative to the rabbit pyrogen test. In a recently peer-reviewed published study, researchers from Charles River’s Microbial Solutions division compared the results of LAL and recombinant bacterial endotoxin tests on 128 contaminated pharmaceutical water samples to see whether the synthetic tests measured up to the traditional LAL.
Recombinant Factor C (rFC), the first enzyme in the endotoxin activation of LAL is touted as a synthetic replacement to LAL. As a single enzyme, rFC reagents do not possess the natural “amplification” of the biological response that is seen in LAL. As such, all of the available rFC reagents rely on fluorogenic substrates for sensitivity. While Fluorescent technology provides the sensitivity for an alternative to the LAL test, it also involves expensive instrumentation.
Recent publications have shown that Factor B plays an important role in not only the activation of the Limulus pro-clotting enzyme but also to the specificity of the endotoxin mediated LAL test. Given this contemporary research and given that the pro-clotting enzyme is responsible for the biological amplification of LAL, Charles River is developing a recombinant product that contains all three of the essential LAL enzymes (Recombinant Factor C, recombinant Factor B and Recombinant Pro-Clotting enzyme).
The purpose of this water study was to compare two FDA-licensed LAL reagents to all of the commercially available recombinant Factor C products, and our own recombinant – full cascade - LAL. The pharmaceutical water samples, if not properly treated, could endanger manufacturing processes, products, and patient safety. Any alternative method to the BET must detect the environmental endotoxins present in pharmaceutical manufacturing facilities.
The Charles River study used samples from pharmaceutical water systems. Previous studies have used purified lipopolysaccharides, clean samples of Water for Injection, and even air samples from non-pharmaceutical sites, none of which can demonstrate true comparison to natural endotoxins found in everyday pharmaceutical work.
Analysis of the study results shows that rFC is inferior to LAL, and it is clear from the data that rFC misses a number of the naturally occurring endotoxins within the provided samples in comparison to natural LAL. The study results show that our fully recombinant LAL performed better than rFC, however it is also inferior to LAL. The results from this study demonstrated that all recombinant products have a propensity to underpredict endotoxin activity when compared to the BET.
As responsible contributors within the biopharmaceutical industry, the quality of the products manufactured, efficacy of the testing, and safety of patients are our main priority. It is our recommendation that LAL remains the gold standard and continue to be used until a suitable, comparable alternative method can be found.
Charles River continues to develop their rLAL and research the propensity for recombinant BET products to underestimate compared to natural LAL.