Endotoxin detection is not just a regulatory requirement; it's a critical factor in ensuring pharmaceutical safety. It's time to discover the transformative power of Recombinant Factor C (rFC) and its journey from being an alternative assay to becoming a compendial method.

Recombinant Factor C (rFC) is recognized as a compendial test in the 27 member states of the European Union (Ph. Eur. Supplement 10.3). Its acceptance and utilization in Europe provide a solid foundation for its broader recognition in the field of endotoxin testing.

In the United States, rFC has long been considered an alternative assay, subject to validation under USP <1225>. However, the status of rFC is now poised for a significant transformation as the US Pharmacopeia prepares to publish a new chapter (USP <86>) that categorizes rFC as a compendial test. This move aligns with USP's commitment to reducing the use of animal-derived materials and represents a major step forward in simplifying and modernizing endotoxin testing.

One of the most compelling aspects of adopting rFC is the validation process, which is far simpler than many assume. The transition from Limulus Amebocyte Lysate (LAL) to rFC can begin with tests on water, raw materials, and closure containers since these tests are said to be “inspectional” in that they do not require permission for change. This isn't just a change; it's an opportunity to gain a competitive edge going forward.  These tests account for 80-90% of pharmaceutical testing, making them the perfect starting point for achieving supply chain assurance and sustainability goals. 

Validation is necessary for any test and for compendial tests is referred to as “verification”.  Verification considers that the method is already validated and concentrates on verifying sample compatibility.  Method validation utilizes purified water to establish method quality attributes (accuracy, precision, ruggedness, etc.).  These attributes have been repeatedly successfully established with rFC as per the manufacturer’s available validation data.  Below is a recommended outline when beginning such testing.  

• Acquire a fluorescent reader compatible with rFC reagents and software.
• Establish Standard Operating Procedures (SOPs) governing various aspects of the rFC test.
• Qualify the reader per the manufacturer's instructions.
• Conduct analyst training.

The validation process for rFC as a pre-compendial method involves method validation as per USP <1225 and sample validation per USP <85>, which closely mirrors LAL's sample verification process.

Compendial chapter USP <86> will remove the necessity of demonstrating method attributes (accuracy, precision, limit of detection, etc.).  Thereby, USP <86> will make the verification expectations on par with current LAL verification performance.   

The Method Validation demonstrates the required quality attributes expected of the Bacterial Endotoxin Test (BET), including accuracy, precision, specificity, linearity, range, detection limit, quantitation limit, and robustness. Specificity is particularly noteworthy, as rFC does not produce false positive reactions with various non-endotoxin substances, such as beta-glucans.

It it important to remember the following points:

• Focus on meeting the requirements of alternative method validation.
• Method validation uses purified water only and is performed once per laboratory.
• Validation should not be viewed as a barrier to implementation, but rather part of the learning process of implementing rFC and gaining lab proficiency.  

• Document item validation for any sample that is not purchased purified water.
• Include a non-inhibitory concentration (NIC) test and an inhibition/enhancement (I/E) test.
• These tests ensure that interference properties that are present can be overcome, establishing the routine test dilution range (between the NIC and MVD).
• Control standard endotoxin/reference standard endotoxin (CSE/RSE) spiked and non-spiked endotoxin sample PPCs are used for validation.

With a qualified reader and SOPs in place, a full validation can be completed in as little as two days, requiring just six rFC assays, involving two operators and three reagent lots.  If desired, request and follow the reagent manufacturer’s validation documentation or allow the reagent manufacturer to perform the method validation in your lab.

Both the FDA and the European Directorate for the Quality of Medicines and Helathcare (EDQM) have recognized the advantages of rFC and its equivalency with LAL. EDQM has published a compendial rFC test chapter (2.6.32), while FDA guidance mentions rFC assays for BET.

Additionally, the USP is in the process of adopting rFC within its regulatory framework, in the upcoming Chapter <86>. This development solidifies rFC's position as the future of endotoxin testing, recognized not only in Europe but also in the U.S. 

Implementing rFC is not just a smart choice; it's the strategic move that will set you apart. Our team is ready to guide you through this transformative journey. In a mere week, you can have the rFC method fully implemented and rigorously validated for routine testing.

RFC offers compatibility with high throughput testing requirements, such as purified water assessments as the reagent are completely stable at room temperature for several hours.  And rFC aligns seamlessly with your sustainability and supply chain assurance goals as a non-animal derived reagent.