Recombinant Factor C Assay

rFC White Paper, “From LAL to rFC”

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Recombinant Factor C Assay Principle

PyroGene^TM rFC Enzymatic Cascade

The PyroGene^TM rFC Assay includes 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 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.

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Recombinant Factor C Regulatory Status

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 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.)

Horseshoe Crab Conservation

At Lonza, we are aware of the importance of the horseshoe crab for its contribution to the safety of drugs and medical devices, but also its ecological role. We committed to protecting the welfare of the horseshoe crab through adopting best practices and actively supporting conservation efforts.

We continue sponsorship of horseshoe crab conservation initiatives developed by the Ecological Research and Development Group (ERDG), a non-profit wildlife conservation organization whose primary focus is the conservation of the world's four horseshoe crab species, to help raise conservation awareness.

PyroGene^TM Validation Timeline

A possible validation scheme is outlined below (one validation can be accomplished in as little as 5 days assuming that the product has been previously validated with a quantitative LAL method).The validation scheme is identical to that which would be needed for any LAL-based method with just the addition of one extra step, ”Validation of Alternative Method.

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It’s Time to Embrace the Alternative

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FDA Approves First Drug Using the recombinant Factor C Assay for Endotoxin Testing

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Regulatory Update

Responses to USP's chapter 1085.1 have been posted on USP's website, including those from Lonza. This is a departure from previous responses provided to USP, as these were always confidential. Some of the responses found on USPs website were clearly marked "Confidential", yet were published.

EP chapter 2.6.32 regarding recombinant Factor C assays became official as of 01 January 2021. This chapter is accompanied by the revised 5.1.10. Guidelines for using the test for bacterial endotoxins, which has been updated to reflect the new status of rFC-based methods and give prerequisites for their deployment by users of the pharmacopoeia.

The Japanese Pharmacopoeia published "Bacterial Endotoxins Test and alternative methods using recombinant protein reagents for endotoxin assay", and the comments period was closed on 30 October 2020.

December 2019

The European Pharmacopoeia (Ph. Eur.) Commission has adopted general chapter 2.6.32 Test for Bacterial Endotoxins using Recombinant Factor C

Lonza is happy to provide our customers with validation support when assessing PyroGene® rFC Assays for use in testing their products. Lonza will provide a detailed protocol that is essentially a "fill in the blanks" document that may be provided to authorities in regulatory submissions. The document follows the requirements found in USP <1225> "Validation of Compendial Methods", and assesses accuracy, range of quantitation, specificity, linearity, precision, and range. These are the same parameters found in ICH Q2B "Validation of Analytical Procedures: Methodology", and are specified as requirements in the 2012 FDA guidance "Pyrogen and Endotoxin Testing: Questions and Answers". These were the same procedures used in Lonza’s Stimulus to Revision article (1) published in the USP’s Pharmacopeial Forum in January 2010. Lonza will also provide expert support for customers that may need technical assistance when validating their products for regulatory filings.

(1) Pharmacopeial Forum Vol. 36(1) [Jan.–Feb. 2010], Stimuli to the Revision Process: A Recombinant Factor C Procedure for the Detection of Gram-negative Bacterial Endotoxin.

An Animal-free Alternative for Endotoxin Testing

the next source for endotoxin testing. . . Let's use it together.

For over 30 years, you have relied on us for our endotoxin expertise. But we know that science is never finished. Our commitment to sustainability drives us forward, motivated by figuring out something that no one has before.

How we address the rise in global populations, personalized medicines and the dependency on horseshoe crabs for endotoxin testing is the next game changer.

With the recombinant Factor C Assay, we can meet the demands of endotoxin testing, protect a natural resource and together, and bring your next medicine to life.

Dependency on Horseshoe Crabs

The supply of the LAL and TAL tests is solely dependent on the horseshoe crab (Limulus polyphemus for the LAL assay or Tachypleus gigas and Tachypleus tridentatus for the TAL assay). With evolutionary ancestors dating back to the Paleozoic era, 540 to 248 million years ago, these living fossils predate humans and dinosaurs. Should anything happen to the population of these crabs, the world’s supply of LAL/TAL-based tests would be in jeopardy. Obtained from the blue blood of the horseshoe crab, Limulus and Tachypleus Amebocyte Lysate (LAL/TAL) reacts to the presence of bacterial endotoxins and has been used for endotoxin testing since its commercialization in the 1970s. LAL/TAL-based assays are the current methods of choice for testing parenteral pharmaceuticals for the presence of bacterial endotoxins (TAL is only used in Asia).

The PyroGene^TM recombinant Factor C Assay is the evolution of the LAL test. Combining 21st century technology with horseshoe crab’s endotoxin sensitive protein, we have developed an equivalent, reliable and sustainable endotoxin detection method for the future.

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.

Worldwide, there is a growing appeal in the pharmaceutical industry to consider recombinant technologies that can help alleviate, and eventually replace, the need for animal resources for endotoxin testing purposes. Adoption of alternative test methods, like PyroGene^TM rFC, are on the rise as these recombinant methods have proven equivalency to LAL-based methods and therefore, companies can feel confident in implementing rFC methods in their QC testing labs.

For a detailed product overview, educational materials, and scientific data about the effectiveness of rFC as a BET assay, please visit the PyroGene^TM rFC Product Page.

PyroGene^TM rFC Assay

Validation Protocol

Validating the PyroGene^TM rFC Assay for your endotoxin testing needs is easy with our protocol.

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Citations List

Recombinant Factor C Assays

A Lonza-curated list of peer-reviewed articles related to recombinant Factor C assays for the detection of bacterial endotoxins.

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We offer a full Validation Protocol that be followed for your convenience. For further information, please submit your Validation Protocol Request below or contact our Scientific Support Team.

The demand for LAL/TAL testing is growing due to a rise in the production of parenterally administered pharmaceuticals (including biologics), all of which will need to undergo endotoxin testing. As more countries become ‘economically developed’, healthcare needs will inevitably increase as well as the number of persons with access to medical care. The amount of LAL/TAL required to keep up with medical and economic advances may become a cause for concern, as the world’s supply of horseshoe crabs is a potentially finite resource, especially if their use if not managed properly. Lonza scientists developed the recombinant Factor C assay to help alleviate the pressures of a growing demand for LAL and TAL. The recombinant Factor C assay does not use horseshoe crab blood as the raw material, therefore, there is less dependence on the animal, which results in less animal utilization.

The 165th European Pharmacopeia (Ph. Eur.) Commission recently 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. After releasing a draft text for comment in November 2018, the European Pharmacopoeia is now taking the next step to make endotoxin testing with recombinant Factor C (rFC) a compendial method in Europe. This marks the first time the recombinant Factor C method, like the PyroGene^TM rFC Assay, will be considered a compendial BET assay by a global regulatory authority. This is also exciting news for users of Lonza’s PyroGene^TM recombinant Factor C assay, as it will allow customers to use rFC without the necessity for an alternative method validation. The new chapter 2.6.32 is announced to be published on 01 July 2020 in Ph. Eur. supplement 10.3 and will be effective as of 01 January 2021. Read the press release.

the next source for endotoxin testing . . .

the next source for endotoxin testing . . .

Recombinant Factor C Assay

rFC White Paper, “From LAL to rFC”

Released in our QC InsiderTM Toolbox

Confidence for Adopting the rFC Method in Your Lab

Data Sheet

PyroGeneTM rFC Enzymatic Cascade

PyroGeneTM Validation Timeline

PyroGeneTM White Paper in our QC InsiderTM Toolbox

FDA Approves First Drug Using the recombinant Factor C Assay for Endotoxin Testing

Regulatory Update

December 2019

The European Pharmacopoeia (Ph. Eur.) Commission has adopted general chapter 2.6.32 Test for Bacterial Endotoxins using Recombinant Factor C

An Animal-free Alternative for Endotoxin Testing

the next source for endotoxin testing. . . Let's use it together.

Dependency on Horseshoe Crabs

PyroGeneTM rFC Assay

Validation Protocol

Citations List

Recombinant Factor C Assays

Released in our QC Insider^TM Toolbox

PyroGene^TM rFC Enzymatic Cascade

PyroGene^TM Validation Timeline

PyroGene^TM White Paper in our QC Insider^TM Toolbox

PyroGene^TM rFC Assay