For more than 40 years, the Limulus Amebocyte Lysate (LAL) test has served as the standard method for bacterial endotoxin Testing (BET). The critical proteins required for endotoxin detection in LAL assays are sourced from amoebocytes-blood cells extracted from horseshoe crabs.Animal-free reagents have been developed to support conservation efforts and the 3Rs (Replacement, Reduction, and Refinement). One such alternative is the recombinant cascade reagent (rCR), which contains three recombinant proteins that replicate the natural enzymatic cascade found in horseshoe crab amoebocytes to detect bacterial endotoxins.This study assessed the equivalency of rCR to FDA-licensed LAL reagents using 563 pharmaceutically relevant samples. Of these, 134 samples were contaminated with natural environmental endotoxin (NEE), allowing for a direct statistical comparison.Results showed that the rCR assays were equivalent in performance to FDA-licensed LAL assays, detecting endotoxin at similar levels under real-world conditions. Equivalency was demonstrated using methods consistent with those outlined in compendial guidance for bacterial endotoxin testing.
Cramer et al. (Thu,) studied this question.