Advanced Recombinant Cascade Reagent PyroSmart NextGen^® for Bacterial Endotoxins Test as Described in the Pharmacopeias

Abstract

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.

Evaluation of Recombinant Cascade Reagent PyroSmart NextGen^® and Limulus Amebocyte Lysate Equivalency in a Plate and Tube Reader for Bacterial Endotoxins Testing

“…With the results from a previous study, the analytical performance and suitability of PyroSmart NextGen ® as an alternative to LAL reagents have been confirmed. 9) Equivalency of PyroSmart NextGen ® and LAL Reagents A large sample size was tested to provide statistical significance of the resulting data. 13) Out of the 102 samples tested, a total of 68 samples tested using the plate reader method and 74 samples tested using the tube reader method had valid PPC recoveries within 50-200% and were therefore included in further data analysis.…”

“…10,11) The ICH guideline M10 Bioanalytical Method Validation and Study Sample Analysis and a previous study were referenced when assessing all acceptance criteria. 9,12) This study included 16 assays tested by two analysts using two tube reader instruments over two days with two reagent lots. Preliminary tube reader testing of PyroSmart NextGen ® demonstrated a sensitivity of 0.001 EU/mL.…”

“…This reagent has previously met the requirements for method validation with a plate reader instrument. 9) For a comprehensive analysis, the performance and analytical characteristics of PyroSmart NextGen ® in a tube reader were evaluated here according to the ICH Q2 guideline and USP <1225>. 10,11) This study is a critical extension of PyroSmart NextGen ® testing because it validates the utilization of a tube reader, with a five-fold sensitivity increase from a detection limit of 0.005 to 0.001 EU/mL.…”

A Demonstration of the Validation Process for Alternative Endotoxin Testing Methods Using PyroSmart NextGen^® Recombinant Cascade Reagent

“…Although specificity for detecting endotoxin in the presence of (1→3)-ß-D-glucan was evaluated in previous studies not shown here, additional analysis when testing one lot of Sodium Citrate for Injection was included to provide an example using a sample matrix other than water. 10) Based on previous data, this testing was performed using the maximum valid dilution (MVD), the MVD/2, and the MVD/5 with corresponding positive product controls (PPCs) at a final concentration in the middle of each standard curve. These sample results were evaluated for suitable precision (repeatability), valid PPC recoveries, and detectable endotoxin.…”

“…A previous study testing samples containing autochthonous endotoxin established the methods for evaluating this equivalency against in-house criteria. 10,11) This study assesses the analytical performance of PyroSmart NextGen ® , Pyrochrome ® (chromogenic LAL), and Pyrotell ® -T (turbidimetric LAL) according to in-house acceptance criteria and directly compares the results to demonstrate equivalency. Analytical performance includes analysis of linearity, accuracy, precision, range, quantitation limit, and specificity according to USP <1225> and the ICH Q2 guideline.…”