Global Pharmacopoeia Harmonization for Modern Pyrogen and Endotoxin Testing

August 14, 2026

Scientific advancements in recent decades have facilitated the transition to new animal-free technologies: the monocyte activation test (MAT), recombinant Factor C (rFC) test, and the recombinant Cascade Reagent (rCR) test, represent leading-edge methodologies in next-generation pyrogen testing. The US Pharmacopeia (USP) has included the use of rCR and rFC testing within its guidelines, whereas only the MAT and rFC tests are included in the European Pharmacopoeia (Ph. Eu.).

Background

The detection of pyrogens is a vital safety measure in the manufacturing of injectable pharmaceuticals. The majority of contaminants are endotoxins that originate from the outer membrane of gram-negative bacteria. They can cause severe fever or toxic shock if administered intravenously. Additionally, non-endotoxin pyrogens also pose a significant threat, comparable to that of endotoxins.

To combat this, the USP first introduced pyrogen testing as a requirement for injectables and medical devices in 1942. During that time, the Rabbit Pyrogen Test (RPT) was the standard method. But, by the 1970s and 1980s, it was discovered that an enzyme, “Factor C,” found in the amebocytes of horseshoe crabs, binds to endotoxins, activating a cascade that results in the production of insoluble clotting proteins. This was used to develop the bacterial endotoxin test (BET) in 1980 as a replacement for pyrogen testing. However, in more recent years, the development of the MAT has been used as an alternative to the RPT¹, and recombinant protein reagents (rFC and rCR) have been used as alternatives to horseshoe crab-derived reagents. The development of these methods is vital for the future of sustainable, animal-free QC testing.

rFC and rCR Testing

The genomes of the horseshoe crabs have recently been used to develop recombinant proteins from the natural LAL cascade, which are key components in rFC and rCR tests. Unlike naturally sourced LAL reagents, these alternative reagents offer advantages such as avoiding lot-to-lot variability and minimizing false positives from the (1→3)-β-D-glucan coagulation pathway. Moreover, the use of recombinant reagents promotes sustainable endotoxin testing, aligning with the replacement, reduction, and refinement principles for animal welfare.²

In response to these advancements, in September 2019, the USP Microbiology Expert Committee proposed edits to the harmonized USP-NF bacterial endotoxins test chapter <85>, which included the addition of recombinant LAL reagents; however, this proposal was rejected. Other proposals also failed, such as the addition of an advisory chapter, <1085.1>, leaving recombinant LAL (rLAL) reagents as alternative methods.

Then in 2022, the USP Microbiology Expert Committee was dissolved and replaced with a new committee. Upon the arrival of this new committee, Fujifilm released the PYROSTAR Neo+ internal validation package to aid in the validation of the method in 2023. In 2024, the USP voted to include rFC and rCR test methods in USP <86>, and by May 2025, it was officially included in USP-NF 2025 Issue 1.³

Regarding the implementation of rCR tests, it is important to note that section 2.6.14 of the Ph. Eur. lists rFC reagents as a compendial method for BETs, and does not specifically address or provide guidance on the use of rCR. As a result, in Europe, the rCR method is viewed as a non-pharmacopeial alternative.⁴

LumiMAT Pyrogen Detection Kit

Human cells are used in the MAT, which allows for greater physiological relevance. Additionally, where LAL only tests for endotoxin contamination, MATs account for contamination from non-endotoxin pyrogens (NEPs) as well.⁵ Most commercially available MAT kits use peripheral blood mononuclear cells (PBMC) for an ELISA to conduct the test. Variations between lots of PBMC is common, and the test’s turnaround time averages two days due to the need for cell culture, ELISA, and multiple pipetting and washing steps.

In contrast, the LumiMAT Pyrogen Detection Kit from FUJIFILM Biosciences detects pyrogens using a novel NF-κB reporter gene assay. The NF-κB reporter gene assay offers several advantages, including freedom from ELISA, ease of handling, and significantly reduced reaction time due to the elimination of the need to wait for IL-6 release. LumiMAT can provide accurate results in less than half the time of traditional MAT kits, utilizing a luminescence readout instead of the traditional ELISA methodology.

The European Directorate for the Quality of Medicines & HealthCare, the Council of Europe, and the European Partnership for Alternative Approaches to Animal Testing collaborated on an international conference: "The Future of Pyrogenicity Testing: Phasing Out the Rabbit Pyrogen Test." The conference's aim was to showcase the European Pharmacopoeia's strategy for removing the RPT from its texts by 2026, streamlining MAT implementation, and identifying shortcomings in RPT eradication.⁶

The FDA acknowledged the potential use of the MAT after product-specific validation in 2009, and later published guidelines that included its possible use for FDA-regulated products like medical devices. The USP’s general chapter on pyrogen testing permits substituting a validated in vitro pyrogen or bacterial endotoxin test for the RPT.⁷

The validation process should include different tests, such as interference screening and method suitability testing, while also ensuring analyst and lab qualifications are validated for the assay.⁸

Conclusion

As regulatory bodies begin to recognize alternative, animal-free pyrogen testing methods, it is vital to understand the difference between such tests. Products such as the LumiMAT Pyrogen Detection Kit and the PYROSTAR Neo+ rCR endotoxin reagent offer sensitive detection of endotoxin and non-endotoxin pyrogens without the use of rabbit or horseshoe crab-derived methodologies.

Learn more about the pyrogen and sterility testing solutions offered by FUJIFILM Biosciences and discover how we are committed to more ethical approaches to science.

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