Recombinant Factor C Assay

The PyroGene^TM Recombinant Factor C Assay is the evolution of endotoxin detection testing. Based on the activation of a recombinant form of Factor C, the first component in the horseshoe crab clotting cascade activated by the presence of endotoxin, the recombinant Factor C assay offers the same reliability as an LAL method – without the use of animal resources. The recombinant Factor C method works through a single enzymatic step compared to the multiple step enzymatic process necessary for LAL assays.

Learn more about our rFC assay by visiting the PyroGene^TM Recombinant Factor C Assay product page where you can find comprehensive details, including a product overview, educational materials, and scientific data about the effectiveness of rFC as a BET assay.

In the PyroGene^TM rFC assay, activated Factor C directly cleaves a fluorogenic substrate, producing a signal that is read using a standard fluorescent plate reader. Due to the high dynamic range of the fluorescent signal, PyroGene^TM rFC delivers a quantitative range of 5.0 EU/ml – 0.005 EU/ml in a single step, with better resolution than conventional kinetic LAL assays.

The PyroGene^TM rFC method is a quantitative assay that is run on a 96-well plate incubated in a microplate reader at 37°C that measures fluorescence with excitation/emission wavelengths at 380/440 nm. In the presence of endotoxin, activated rFC will cleave the fluorogenic substrate, causing the solution to fluoresce.

The graph on the left illustrates the enzymatic cascade of the recombinant Factor C assay compared to the enzymatic activation pathway of traditional LAL assays, like the kinetic chromogenic and kinetic turbidimetric assays.

Accepted by several global regulatory authorities including the FDA and the European Pharmacopeia, the recombinant Factor C method is a recognized, comparable endotoxin detection method to LAL-based assays. USP 28-NF 33 General Notices states that alternative testing methods, such as recombinant Factor C (rFC) can be used if the alternative method provides advantages in accuracy, sensitivity, precision, selectivity, or adaptation to automation.

• In Dec 2019, The 165th European Pharmacopeia (Ph. Eur.) Commission announced that it expects to include Chapter 2.6.32, Test for bacterial endotoxins using recombinant Factor C, as one of the 4 general chapters to be adopted in its next version, effective January 1, 2021
• The United States Pharmacopeia (USP), via their official journal, Pharmacopeial Forum (PF), has announced plans to develop a draft chapter <85> Bacterial Endotoxins Test, which will include the addition of recombinant Factor C assays
• In Jan 2019, recombinant Factor C (rFC) has been listed and described as a new compendia method for bacterial endotoxin testing in the Chinese Pharmacopeia, following the EP, JP, and USP. The 4th version of the Chinese Pharmacopeia will be effective in 2020
• In December Dec 2018, The European Pharmacopeia released a draft of their new compendial Chapter 2.6.32 that is dedicated to the recombinant Factor C method
• In September 2018,the U.S. Food and Drug Administration approved the first drug released using a recombinant method for endotoxin testing instead of traditional LAL-based methods for a monoclonal antibody drug treatment for the prevention of migraines in adults. Read Press Release
• Chapter 5.1.10 officially became effective on July 1st, 2016. 510(K) submissions have been approved by the FDA using PyroGeneTM rFC assay as a final release test. Lonza has also submitted a comprehensive FDA Master File
• In July 2015, rFC became officially recognized by the European Pharmacopoeia (Ph. Eur.) as an alternative endotoxin detection methodology to the LAL and Rabbit Pyrogen Tests in the new draft of Chapter 5.1.10*
• In June 2012 the FDA issued the document “Guidance for Industry Pyrogen and Endotoxins Testing: Questions and Answers” which allows for the use of a recombinant Factor C based assay as an alternative to LAL-based assays*

*The rFC assay will still be considered an “Alternative Test”, subject to the validation requirements of USP <1225> or ICH Q2B.  Regulatory authorities will accept the test results of the recombinant Factor C assay, but a validation study must be performed for each product that will be tested using this method. (Validation studies are used to compare the alternative and compendial method, and verify the equivalence between the two methods of the assay.  Post-validation, it is necessary to follow up with the appropriate regulatory filing for the drug product or device.)