Animal-free alternative method for endotoxin testing: introduction of Pyrostar™ Neo

Endotoxins are powerful pyrogens, which can cause severe fever, inflammation, and even septic shock if they gain access to the bloodstream. To prevent or promptly detect endotoxin contamination, both parenterally administered pharmaceutical products and their raw materials are subjected to strict endotoxin testing.

The Limulus amebocyte lysate (LAL) assay has been established as a gold standard for endotoxin testing. It is characterized by high sensitivity and specificity and can be performed as a qualitative or a quantitative test.

However, production of the LAL reagent requires capture and bleeding of horseshoe crabs, which can be a significant source of injury and even potential mortality for the crabs. Due to the high number of horseshoe crabs undergoing this procedure each year, its negative environmental impact is pronounced.

Therefore, animal-free alternatives of the LAL assay that do not require bleeding of horseshoe crabs, and that comply with the 3Rs principles (replacement, reduction, and refinement of animal testing) are needed.

PyrostarTM Neo as a synthetic alternative of the LAL assay

FUJIFILM Wako has developed a three-factor recombinant, 100% animal-free, fully synthetic alternative of the LAL assay named Pyrostar™ Neo. It contains three recombinant factors (Factor C, Factor B, and a proclotting enzyme) and mimics the activation of the coagulation cascade observed in the LAL assay. Moreover, Pyrostar™ Neo reagent contains a pNA chromogenic group, enabling detection of the assay results on an absorbance plate reader using a colorimetric method.

Types of recombinant reagents for endotoxin testing

Recombinant reagents for endotoxin tests can be two types: recombinant cascade reagents (rCRs) and recombinant Factor C (rFC) reagents. rFC reagents include only one of the enzymes of the clotting cascade (Factor C) activated in the LAL assay , whereas formulations of rCRs include Factor C, Factor B, and a proclotting enzyme. In addition, the rFC assay is a fluorometric test, whereas rCR assays are chromogenic. To sum it up, Pyrostar™ Neo is a chromogenic rCR assay and is a sustainable and animal-free alternative.

Accuracy, sensitivity, specificity, and stability of PYROSTAR™ Neo

PYROSTAR™ Neo is a sensitive and accurate reagent with a detection range 0.001–50 EU/mL. Moreover, it is endotoxin-specific, eliminating the risk of false positive results caused by the presence of (1-->3) ß-D-glucan. 

In addition, PYROSTAR™ Neo is characterized by high stability. After its reconstitution, the PYROSTAR™ Neo reagent is stable for 4 hours at 2–8°C or for up to 14 days at -30°C, which is especially beneficial for low test-amount users in 1 day. 

An excellent choice for pharmaceutical applications due to its sustainable nature

The Pyrostar™ Neo endotoxin testoffers clear ethical and environmental advantages over the traditional LAL test, including its compliance with the 3Rs principles of replacement, reduction, and refinement of animal testing. Thus, it promotes ethical and sustainable use of animal testing and helps safeguard animal welfare. After its full validation according to compendial requirements, Pyrostar™ Neo could be considered a suitable alternative for the detection of endotoxin contamination based on a comparable accuracy like the traditional LAL reagent. 

A promising alternative for endotoxin testing of pharmaceuticals and medical devices

Overall, Pyrostar™ Neo provides a reliable and sustainable solution for endotoxin detection in pharmaceuticals and medical devices. Moreover, it can accurately detect endotoxin levels in both raw materials and end products.