Multi-center study of the recombinant cascade reagent (kinetic chromogenic assay) as an alternative method for bacterial endotoxin testing: Method validation, product suitability, and consistency evaluation with limulus amebocyte lysate.

Given the scarcity of global Limulus resources, developing animal-free alternatives for bacterial endotoxin testing is imperative. This study evaluated the Recombinant Cascade Reagent (rCR) kinetic chromogenic assay as a sustainable alternative to traditional Limulus Amebocyte Lysate (LAL). Eleven laboratories collaborated to conduct a comprehensive validation of five commercial rCRs, assessing methodological performance, product suitability across 34 diverse pharmaceutical products (105 batches), and consistency with LAL using 82 naturally contaminated water samples. Results showed that rCRs met pharmacopoeial acceptance criteria for accuracy (50%-200% recovery), precision (RSD < 32%), and linearity (|r| > 0.980), with a limit of detection at 0.005 EU/mL. Interference testing demonstrated a 97.1% overall product suitability rate, including for complex biologics. Consistency evaluation revealed a significant positive correlation between rCR and LAL (Spearman's r > 0.7889), with no statistically significant differences in endotoxin quantification (Wilcoxon p > 0.05). Furthermore, rCR exhibited diagnostic accuracy equivalent to or higher than LAL (Youden's Index: 0.811-0.904 vs. baseline 0.535). In conclusion, the rCR kinetic chromogenic assay demonstrates excellent precision, broad applicability, and high consistency with LAL, proving to be a reliable and standardized alternative for industrial endotoxin testing.