Skip to main content

Bacterial Endotoxin Test (BET): A USP Comparability Study of Recombinant Reagents (Recombinant Factor C and Recombinant Cascade) to Lysate Reagents (LAL)

Type of Posting: General Announcement
Posting Date: 25–Jun–2021

USP is planning to conduct a study to collect data on the comparison of recombinant reagents and lysate reagents described in <85> Bacterial Endotoxins Test

  1. Purpose:

    To compare levels of endotoxins activity arising from autochthonous Gram-negative bacteria present in pharmaceutical (deionized) water samples as analyzed by both the current commercially distributed lysate reagents and recombinant reagents.   

  2. Reagents used in this study:

    Four (4) recombinant reagents (candidate reagents/methods)

    Four (4) lysate reagents

    Note: For the purposes of this study, the term, “recombinant reagent” is defined as a non-lysate formulation containing one or more cloned zymogens from the Horseshoe Crab clotting cascade, and the term, “lysate” is defined as reagents sourced from the amebocytes of live Horseshoe crabs. 

  3. Samples for study:

    Water sampled post-deionization but prior to depyrogenation processes is the test article for this protocol. If an RO membrane is included between the deionization and distillation, the sample will be taken prior to the RO membrane.

  4. Number of Samples and Statistical methods:

    To estimate the appropriate samples size for the primary objective, a sample size of 90, for each reagent, with a non-inferiority margin of 10% will be used. If any sample has endotoxin values below the limit of detection for all methods, it will be removed from statistical analysis.

  5. Study Considerations:
    • Calibration curves and positive product controls (PPC) will be prepared from the USP reference standard endotoxin (RSE).
    • Calibration curve range will be 5.0, 0.5, 0.05 EU/ml for all methods (LLOQ = 0.05 EU/mL).  
    • Positive Product Control (PPC) = 0.5 EU/mL
    • Standards and samples will be tested in duplicate. Samples will be tested undiluted and at a 1:10 dilution.
    • The package inserts for the various test methods will be followed, using glucan blocking reagents as appropriate for each of the lysate reagents. 
    • Each sample will be assayed using each method simultaneously (within a four-hour period).

  6.  Acceptance Criteria:
    • Standard curve must be linear (|r| > 0.980).
    • Negative controls must not react before the 0.05 EU/mL standard.
    • PPC recovered within the range of 50% - 200% of the nominal spike.
    • To be included in the data set, samples must contain quantifiable endotoxin activity (above LLOQ).
    • If the one-sided 95% confidence lower bound exceed 50% per the analysis, non-inferiority of recombinant cascade and rFC reagents relative to LAL will be declared.
    • Data will be analyzed for comparability by the USP statistician and will be reviewed and approved by the USP Expert Committee, Microbiology General Chapters.

If any stakeholders are interested in participating by providing suitable water samples or if you have any questions, please contact Leslie Furr, Senior Scientist I (