Recombinant Factor C method, benefits and validation

By
  • Paula Paci
  • Sara Hully
  • Fabian Vandermeers
  • Géry Van Vyncht
  • Arnaud Delobel
Date
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Pyrogenic substances in pharmaceutical products can induce severe and potentially life‑threatening fever reactions, making endotoxin detection a critical step for market access. Endotoxins from Gram‑negative bacteria are among the most prevalent and quantifiable pyrogens, traditionally detected using the Limulus Amebocyte Lysate (LAL) assay. However, this method presents several limitations, including animal dependency, variability between batches, and susceptibility to interferences such as β‑glucans. The recombinant Factor C (rFC) method offers an alternative by using a single enzymatic reaction and eliminating animal‑derived reagents while maintaining sensitivity and specificity. This application note details the implementation, validation and performance of the rFC assay for endotoxin testing.

The full technical document is available below for download.

KEY HIGHLIGHTS OF THE METHOD

  • rFC is a single‑step enzymatic fluorescent assay based on recombinant Factor C, unlike the multi‑step LAL cascade
  • Detection range defined by a standard curve from 0.005 to 5 EU/mL
  • Quantification limit established at 0.05 EU/mL and detection limit at 0.005 EU/mL
  • No interference from β‑glucans due to the absence of Factor G pathway
  • Demonstrated linearity (R² up to 0.99), precision (repeatability ≤ 12%), and acceptable recovery (50–200%)

HOW THIS SUPPORTS YOUR PROGRAMME?

The recombinant Factor C method provides a validated alternative for endotoxin detection aligned with current pharmacopoeial expectations. Its implementation supports laboratories seeking reliable, sustainable and interference‑resistant analytical approaches.

  • Enables endotoxin detection without animal‑derived reagents, supporting sustainable laboratory practices
  • Reduces risk of false positives by eliminating β‑glucan interference
  • Supports routine quality control testing after product‑specific validation in GMP environments
  • Compatible with regulatory frameworks (Ph. Eur. 2.6.32, USP <1225> validation requirements)
  • Provides reliable performance across different product types, including biologics and complex formulations

WHERE THIS METHOD APPLIES?

  • Routine endotoxin testing in quality control laboratories after validation
  • Analysis of pharmaceutical products including liposome‑based formulations and monoclonal antibodies
  • Implementation in GMP environments for European and US market requirements

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