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What is PyroGene® rFC Assay?

PyroGene® rFC Assay uses a recombinant version of Factor C—the endotoxin sensitive enzyme found in horseshoe crab blood—to detect lipopolysaccharides from Gram negative bacteria.

PyroGene® rFC Assay contains no horseshoe crab blood components. The assay responds specifically to endotoxin, reducing variability and eliminating reliance on biological lysate supply chains.

Reasons to select rFC Assay

Recombinant Factor C testing has been adopted by many manufacturers as part of a risk based evolution of endotoxin control. Organizations select PyroGene® rFC Assay for its:

  • Specificity: rFC responds only to endotoxin, avoiding false positives from (1→3)-β-D-glucans
  • Supply security: No dependence on seasonal or biological harvesting
  • Sustainability: Supports horseshoe blood-free testing strategies aligned with ESG goals

PyroGene® rFC Assay enables QC teams to modernize endotoxin testing while maintaining continuity with established endotoxin limits and specifications.

PyroGene® rFC Assay – Key technical characteristics

  • Assay type: Fluorogenic recombinant Factor C
  • Detection target: Bacterial endotoxin (LPS)
  • Quantitative range: 0.005–5.0 EU/mL
  • Format: 96-well microplate
  • Readout: Fluorescence (Ex/Em 380/440 nm)
  • Enzymatic steps: Single-step activation

How does PyroGene® rFC Assay work?

PyroGene® rFC Assay leverages a fluorogenic rFC assay format:

1. Endotoxin activates recombinant Factor C

2. Activated rFC cleaves a fluorogenic substrate

3. Fluorescence intensity is proportional to endotoxin concentration

Because Factor C is the first step in the natural endotoxin response cascade, PyroGene® rFC Assay delivers direct, mechanism based detection without downstream components that may introduce variability.

PyroGene® rFC Assay principle

The PyroGene® rFC Assay contains the rFC enzyme cloned from the horseshoe crab. When endotoxin binds to recombinant Factor C, the activated rFC enzyme cleaves a synthetic fluorogenic substrate causing the solution to fluoresce. The recombinant Factor C assay works through a single enzymatic step and offers the same reliability as a LAL method – without the use of horseshoe crabs.

The PyroGene® rFC method is a quantitative assay that is run on a 96-well plate incubated in a microplate reader, like the Nebula multimode reader, at 37°C that measures fluorescence with excitation/emission wavelengths at 380/440 nm. Due to the high dynamic range of the fluorescent signal, PyroGene® rFC Assay 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.

How PyroGene® rFC Assay compares to LAL methods

PyroGene® rFC Assay is functionally aligned with compendial endotoxin testing expectations while offering technical advantages:

  • Comparable sensitivity and quantitation ranges to LAL
  • Improved specificity due to single analyte focus
  • Reduced risk of assay interference
  • Greater control over raw material consistency

For many laboratories, PyroGene® rFC Assay is implemented alongside LAL during validation, or used selectively in defined applications as part of a structured transition strategy.

Response to different endotoxin sources

Endotoxin potency of four purified lipopolysaccharide preparations as tested by multiple methods. The results of a comparison between the responses of the rFC, kinetic chromogenic LAL and kinetic turbidimetric LAL photometric methods to different sources of purified endotoxin. The data demonstrates that the rFC method can recognize endotoxin from different source materials similar to other photometric procedures, demonstrating comparability to the LAL-based methods.

PyroGene® rFC Assay and endotoxin specificity

The PyroGene® rFC Assay does not react with glucans, thereby reducing false-positive results. Comparison of glucan activity between kinetic chromogenic LAL and rFC. The false positive signal from the LAL assay is reduced in the presence of a glucan blocker. The rFC assay is endotoxin specific.

Is PyroGene® rFC Assay accepted by global regulators?

Yes. Recombinant Factor C is recognized in major pharmacopeias, including:

  • European Pharmacopoeia (Ph. Eur. 2.6.32)
  • USP <86> (as an alternative method)
  • JP harmonization pathways

PyroGene® rFC Assay is used today in GMP environments following method validation, comparability studies, and documented risk assessments—consistent with regulatory expectations for alternative methods.

   Learn more about USP <86> guidance   

Frequently asked questions

Is PyroGene® rFC Assay a replacement for LAL?

Yes, PyroGene® rFC Assay can replace LAL in validated applications. Many companies adopt it as a complementary or phased approach.

How does the PyroGene® rFc Assay work? What applications is it most suitable for?

The PyroGene® rFC Assay uses a recombinant form of Factor C that is found in every other endotoxin detection reagent (see Figure 1 below). It directly cleaves a fluorogenic substrate to yield light upon excitation, eliminating the need for the signal amplification steps found in classical assays which are subject to interferences. The PyroGene® rFC Assay works in almost every application that our classical LAL reagents are used in, with the exception of products that are fluorescent.
graph showing how the different endotoxin testing assays work

Does PyroGene® rFC Assay detect non-endotoxin pyrogens?

No. PyroGene® rFC Assay is specific to endotoxin, consistent with endotoxin specific pharmacopeial testing.

What makes Lonza an advanced solution when compared to other options in the market?

The PyroGene® rFC Assay is a liquid preparation and as such, needs no reconstitution step. You simply mix the volume of components you need to test your samples, greatly reducing wasted reagents. This is ideal in high-volume or automated laboratories.

Is method validation required?

Yes. Like any alternative method, PyroGene® rFC Assay requires method validation and, where applicable, comparability to existing assays.

How long will it take to validate Pyrogene® rFC Assay?

A possible validation scheme is outlined in the image below. The validation scheme is identical to the validation scheme for any LAL-based method with just the addition of one extra step, ”Validation of alternative method'.
We offer a full validation protocol for your convenience. For further information, please submit your validation protocol request or contact our scientific support team for a free consultation with our Subject Matter Expert.
image indicating different steps and timelines for validation of Pyrogene rFC Assay

Can PyroGene® rFC Assay be automated?

Yes. The assay is compatible with standard fluorescence based microplate readers and QC automation workflows.

Is PyroGene® rFC Assay suitable for GMP use?

Yes, when implemented within validated methods and quality systems.

What additional equipment/ instrumentation is needed to transition to PyroGene® rFC Assay?

PyroGene® rFC Assay is compatible with standard fluorescence-based microplate readers that meet assay performance requirements, including —but not limited to— Lonza’s Nebula® Multimode Reader.

How does PyroGene® rFC Assay fit Into USP <85>–aligned endotoxin control strategies?

PyroGene® rFC Assay supports endotoxin testing strategies aligned with USP <85> provisions for alternative methods. As a recombinant Factor C (rFC) endotoxin assay, manufacturers commonly implement PyroGene® rFC Assay within established quality systems without changing endotoxin limits, specifications, or acceptance criteria defined under USP <85>. By providing endotoxin specific detection with improved consistency and control, PyroGene® rFC Assay enables QC laboratories to modernize endotoxin testing while maintaining regulatory intent and compliance with pharmacopeial expectations.

Where is PyroGene® rFC Assay used in pharma quality control?

PyroGene® rFC Assay is commonly applied in:
  • Pharmaceutical water systems (PW, WFI)
  • Raw material testing
  • In process samples
  • Finished product release (where justified)

What makes PyroGene® rFC Assay a leading rFC solution?

The recombinant Factor C method is recognized by several global regulatory authorities including the FDA, and the European Pharmacopoeia. It is a comparable endotoxin detection method to LAL-based assays. PyroGene® rFC Assay is considered a well-established rFC assay, supported by:
  • Longstanding commercial availability
  • Broad customer adoption across pharma and biotech
  • Extensive validation precedence
  • Integration into established QC workflows

Its technical robustness and real world usage history distinguish PyroGene® rFC Assay from newer or less proven recombinant assays.

Resources

PyroGene® recombinant Factor C Assay – Endotoxin testing: It's time to embrace the 'alternative'

The LAL assays most commonly used tests in QC micro labs. While the natural resource of lysate have provided a powerful tool to date, there are a number of reasons why the industry can no longer afford to rely on the horseshoe crabs. This white paper addresses the growing demand on natural resource, validated alternatives to it, and benefits of the synthetic version of Factor C.

Read article

PyroGene® rFC Assay validation protocol

According to USP, the rFC assay is 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.Validating the PyroGene® rFC Assay for your endotoxin testing needs is easy with our protocol.

Request validation protocol

Evaluating recombinant reagents

As the industry continues shifting toward sustainable, animal free endotoxin and microbial detection solutions, QC microbiology teams are increasingly asked to evaluate recombinant reagents for use in regulated environments. Yet, with diverse rFC reagent formats, manufacturers, and performance claims varying widely, many labs are left asking the same questions: Where do I start? What should I test? And how do I know whether a recombinant reagent is truly suitable for my process?

In this webinar, Puja Sawhney will share a first hand look at a structured evaluation study designed to compare several recombinant reagent options currently available on the market. Attendees will gain a clear, science driven understanding of how the study was designed, which variables were controlled, and what practical considerations shaped the experimental approach—from sample selection and matrix challenges to sensitivity expectations and data quality criteria.

View webinar