Today, USP <85> continues to serve as the gold standard for Bacterial Endotoxin Testing (BET), relying exclusively on Limulus Amebocyte Lysate (LAL) derived from the blood of the Atlantic Horseshoe Crab (Limulus Polyphemus). While detection methods varied, the reagent initiating the cascade reaction was always LAL. This dependence on a single-source, regionally limited raw material raised concerns about sustainability and supply chain reliability.
LAL is extracted from the horseshoe crab’s blood, primarily sourced from the Northeastern United States. However, environmental challenges such as overharvesting (for bait), habitat disturbance, climate change, and pollution threaten the crab population. These factors jeopardize the consistent availability and performance of LAL, posing risks to the integrity of BET results.
In response, conservation efforts have emerged, including regulatory oversight, habitat protection, and aquaculture initiatives. Simultaneously, the pharmaceutical industry has embraced the Three R’s Principle—Replacement, Reduction, and Refinement—by developing non-animal dependent reagents with comparable efficacy. Although recombinant alternatives have existed for over two decades, compendial recognition was only recently achieved.
In November 2024, USP announced the publication of USP <86>, officially released in May, introducing recombinant reagents for BET. This marks a significant milestone, as both USP <85> and USP <86> are now approved for endotoxin testing.
USP <86> features two recombinant reagents: Recombinant Cascade Reagent (rCR) and Recombinant Factor C (rFC). These are genetically engineered using recombinant DNA to produce the necessary protein complexes for endotoxin detection. The rCR pathway mirrors the kinetic chromogenic LAL method, incorporating Factor C, Factor B, and a pro-clotting enzyme. In contrast, rFC utilizes only Factor C and a fluorogenic substrate, streamlining the detection process.
The rCR reagent offers a seamless transition from LAL, requiring minimal method modification. It also eliminates β-glucan interference, enhances robustness, and reduces lot-to-lot variability. Meanwhile, rFC, though structurally distinct, promises faster reaction times due to its simplified pathway and fluorescence-based detection. The adoption of USP <86> reflects growing confidence in recombinant technology to deliver reliable, sustainable BET solutions. It addresses key concerns:
REPLACE. Substitutes animal-derived LAL with recombinant reagents (rCR and rFC).
REDUCE. Expands market options, decreasing reliance on LAL.
REFINE. Minimizes animal impact by offering non-animal alternatives.
Moreover, recombinant reagents mitigate supply chain vulnerabilities and improve consistency. Despite industry hesitation to shift from a 40-year reliance on LAL, USP recognition and ongoing research are expected to bolster confidence in USP <86>. In time, recombinant technology may not only match but surpass LAL usage and potentially replace it entirely.
