Recombinant animal-free reagents in the bacterial endotoxins test

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Introduction

USP <85>, “Bacterial Endotoxins Test”, contains several test methods used to detect and quantify the activity of endotoxins. USP <85> utilizes reagents naturally sourced from horseshoe crabs, either Limulus polyphemus or Tachypleus tridentatus.

Alternative, non animal-based reagents are produced using the recombinant DNA technology and use one or more cloned zymogen proteases that comprise all of or part of the natural lysate reaction cascade.

Two types of recombinant reagents are currently available: Recombinant Factor C (rFC) and Cascade Reagents (rCR).

Recombinant Factor C (rFC) reagents contain only the recombinant Factor C constituent of the clotting cascade and the test methodology for using rCR is an endpoint fluorometric tests, (see USP <85>, Photometric Quantitative Techniques, Chromogenic technique). Recombinant Cascade Reagents (rCR) include Factor C, Factor B and the proclotting enzyme in their formulations. The test methodology for using rCR is the same as described in USP <85>, under Photometric quantitative (kinetic chromogenic) techniques,

Although the instructions for performing the recombinant methods are essentially identical to the chromogenic endpoint and kinetic methods described in <85>, the reagents are not identical to reagents prepared from horseshoe crab hemolymph. Therefore, endotoxins testing using recombinant reagents are considered to be alternative methods and require validation and demonstration of comparability with the official method.

Validation

A bridging and documented formal study should be performed to demonstrate that the performance characteristics of the analytical methods using the recombinant reagents comply with requirements of USP <1225>, specifically sensitivity, specificity, linearity, ruggedness and robustness.

Comparability

Given the complexity of the lysate prepared from disrupted amebocyte granules, comparability of the recombinant reagents to naturally sourced lysate using endotoxins from autochthonous manufacturing sources is of particular importance. Comparability of recombinant reagents should therefore demonstrate equivalency of results per USP <1223>. It is recommended to compare test results using products that contain known levels of measurable endotoxins activity from a source that could reasonably be expected to contaminate the product.

The following suggestions are provided to minimize variables that may affect the comparability protocol:

Given that the recombinant factor C reagent, have no Factor G pathway, the use of a glucan blocker for the lysate reagent is recommended.
Control standard endotoxins (CSE) are secondary calibration analytes which may be derived from different strains of E. coli and formulated differently among reagent suppliers. The use of one manufacturer’s CSE with another’s reagent may result in a different potency determination that could influence the comparability study outcome (USP <1085>). It is suggested that comparability studies employ the USP RSE for calibration curves and Positive Product Control (PPC) in order to eliminate any effects that an unmatched combination of reagent lot/CSE lot may have on the test result.
An example of acceptance criteria of measured endotoxins activity using recombinant and naturally derived lysates might be the following: The measured activity of a sample containing endotoxins using a recombinant reagent method should fall within 50%-200% of the measured activity in the same sample tested using natural lysate as described in <85>. A sample calculation is provided below for a single sample where endotoxins from autochthonous sources are measured at 4.7 EU/mL using the compendial lysate method and 5.3 EU/mL using the recombinant reagent.

Relative Recovery = [(5.3 EU/mL) ÷ (4.7 EU/mL)] x 100 = 113% recovery

Suitability

Suitability of the recombinant reagent method(s) with the material under test is demonstrated t per USP <85> “Test for Interfering Factors” (see below.)

Preparatory testing and general notes

Apparatus: Reference USP <85>
Reagents and Test Solutions: Reference USP <85> except for the following:
Recombinant Reagent: Follow the manufacturer’s instructions for storage, reconstitution and use.
Preparation of Drug/Biological Product Solutions and Medical Device Extracts: Reference USP <85>, USP <161>, USP <1085>
Determination of Maximum Valid Dilution, Endotoxin Limit and Concentration of Sample Solution: Reference USP <85>, USP <161>
Preparatory Testing (assurance of criteria for the calibration curve): Reference <85>
- rFC Assay: Reference <85>, “Endpoint Chromogenic Technique”
- rCR Assay: Reference <85>, “Kinetic Chromogenic Technique”

Test procedure an interpretation

For rFC and rCR routine test procedures, and interpretation of results follow <85>, for the endpoint chromogenic technique and kinetic chromogenic technique, respectively can be utilized.