The bacterial endotoxins test (BET) is a method of detection and quantification of bacterial endotoxin in injectable drugs and medical devices using amoebocyte lysate reagents sourced from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). Three manufacturers have launched three different types of recombinant reagents, and recently the United States, European and Japanese Pharmacopeias have been evaluating the equivalency of these reagents to natural amoebocyte lysate reagents. Several studies suggested that the recombinant reagents are very similar to amoebocyte lysate reagents, however there is potential for improvement. Previous reports indicate that recombinant reagents have two issues: extremely low endotoxin potency determination for Helicobacter pylori GU2 and high levels of interference from Heparin Calcium impacting endotoxin recovery. A new recombinant cascade reagent (rCR), PyroSmart NextGen ® , recently introduced to the market has been developed to solve these issues. PyroSmart NextGen ® has demonstrated higher reactivity to H. pylori GU2 and a lower level of interference from Heparin Calcium than other existing recombinant reagents. Additionally, the analytical capability and suitability of PyroSmart NextGen ® has been demonstrated when applied to the BET as described in the US, European and Japanese pharmacopeias. PyroSmart NextGen ® has also shown comparability to amoebocyte lysate reagents by demonstrating its ability to detect autochthonous endotoxin in water and in Escherichia coli culture. Overall, this study has verified that the rCR, PyroSmart NextGen ® is a suitable alternative to amoebocyte lysate reagents.

PyroSmart NextGen ® is a recombinant cascade reagent (rCR) for the detection and quantification of bacterial endotoxins developed using cloned genes derived from the Limulus polyphemus horseshoe crab genome. Requirements for use of this alternative reagent include analysis of analytical performance, method suitability, and test result equivalency to Limulus amoebocyte lysate (LAL) reagents used in the compendial Bacterial Endotoxins Test (BET). The plate reader evaluation has been expanded to address two long-standing user preferences, with the inclusion of the tube reader method increasing the sensitivity of endotoxin detection from 0.005 EU/mL to 0.001 EU/mL. The utilization of PyroSmart NextGen ® with the two different instrument types also allows for a more comprehensive equivalency analysis. Furthermore, the comparison results demonstrate that PyroSmart NextGen ® detects equivalent levels of autochthonous endotoxin in water samples when compared to LAL reagents. Overall, this study provides the first large-scale example of equivalency analysis utilizing a robust rCR and has verified that PyroSmart NextGen ® meets the expectations for alternative reagents.