Overview Of Regulations By Different Pharmacopoeia and the Harmonization Efforts For The New Generation Pyrogen And Endotoxin Tests (rFC, rCR, and MAT)

Published on 1st July 2025

Scientific advancements in recent decades have facilitated the transition to new animal-free technologies MAT, rFC, and rCR represent leading-edge methodologies in next-generation pyrogen testing. The US Pharmacopeia has included the use of rCR, RFC and MAT within their guideline. However, European Pharmacopeia has included only MAT and rFC within its guidelines.

 

The detection of pyrogens is a vital safety measure in the manufacturing of injectable pharmaceuticals. These contaminants, categorized as endotoxins and found in the outer membrane of gram-negative bacteria's cell wall, can cause severe fever or toxic shock if administered intravenously. Importantly, non-endotoxin pyrogens also pose a significant threat, comparable to that of endotoxins. 

From the 1940s to the 1970s, the pharmaceutical industry relied on rabbits to detect endotoxins, leading to the death of large number of animals. [1] 

However, in the 1970s, a new approach to endotoxin detection emerged, utilizing the hemolymph of horseshoe crabs. The Limulus Amebocyte Lysate (LAL) test is based on the interaction between amebocytes and bacterial contaminants. This method is widely recognized as the international standard for endotoxin detection. In recent years, demand for sustainable testing methodologies has been a growing trend in all scientific fields. FUJIFILM has significantly increased its dedication to the protection of these remarkable animals through collaborations with various other ecological conservation groups and organizations. This encompasses supporting the Horseshoe Crab Conservation Fund, which is dedicated to the long-term preservation of these crabs via community initiatives and educational outreach.

Overview Of Regulations By Different Pharmacopoeia EN timeline

Recombinant Factor C (rFC) and Recombinant Cascade Reagent (rCR) 

The genome of the horseshoe crab has been recently used to develop recombinant proteins from the natural LAL cascade, which are key components in rFC and rCR reagents (e.g., PYROSTAR™ Neo+). 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. [4] 

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 to the chapter; however, this proposal was rejected. Other proposals failed, such as the addition of an advisory chapter, <1085.1>, leaving recombinant LAL (rLAL) reagents as alternative methods. In 2022, the USP Microbiology Expect Committee was dissolved, with a new committee chosen that reconvened in 2023. Upon the arrival of a new expert committee in 2023, FUJIFILM Wako released the PYROSTARTM Neo+ internal validation package to aid validation of the method. Later that year, chapter <86>, which includes rFC and rCR reagents, was drafted by the USP committee. The USP voted in July 2024 to include USP <86> in the 2025 publication after a public comment period. In May 2025, chapter <86> was officially included in USP-NF 2025 Issue 1. [5] 

Regarding the implementation of recombinant cascade reagents (rCR), it is important to note that the European Pharmacopoeia section 2.6.32, which lists rFC reagents as a compendial method for BET, does not specifically address or provide guidance on the use of rCR reagents. As a result, in Europe, the rCR method is viewed as a non-pharmacopeial alternative. [6]

 

Monocyte Activation Test (MAT) 

Human cells are used in the MAT, which allows for greater physiological relevance. Additionally, where LAL only tests for endotoxin contamination, MAT accounts for contamination by non-endotoxin pyrogens (NEPs) as well. [7] 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, FUJIFILM’s LumiMAT™ Pyrogen Detection Kit 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 RPT from its texts by 2026, streamlining MAT implementation, and identifying shortcomings in RPT eradication. [8] 

The FDA acknowledged the potential use of the MAT after product-specific validation in 2009, later publishing guidelines in 2009 and 2012 that included its possible use for FDA-regulated products like medical devices, provided validation. The U.S. Pharmacopeia's General Chapter on Pyrogens permits substituting a validated in vitro pyrogen or bacterial endotoxin test for the rabbit pyrogen test (RPT), as per USP 2018. [9] 

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. [10]


References:

  1. HealthCare, E.D.f.t.Q.o.M., Ph. Eur. bids adieu to rabbit pyrogen test in its monographs. 2024.
  2. Levin, J. and F.B. Bang, Clottable protein in Limulus; its localization and kinetics of its coagulation by endotoxin. Thromb Diath Haemorrh, 1968. 19(1): p. 186-97.
  3. Pharmacopoeia, U., US Pharmacopoeia—National Formulary [USP 40 NF 35]. vol. 1. Rockville, Md, United States Pharmacopeial Convention. 2017, Inc.
  4. Kelley, M., et al., A Demonstration of the Validation Process for Alternative Endotoxin Testing Methods Using PyroSmart NextGen® Recombinant Cascade Reagent. BPB Reports, 2023. 6: p. 68-75.
  5. Rockville, USP provides guidelines for Recombinant Factor C (rFC) a non-animal-derived reagent critical to development of vaccines and other sterile pharmaceutical products. usp, 2020.
  6. Carmen Marín Delgado de Robles (Roche Basel-Kaiseraugst), D.L.S.G., Evelyn Der (Genentech), Endotoxin testing: the international regulatory landscape. European Pharmaceutical Review, 2024.
  7. Carson, D., et al., Development of a Monocyte Activation Test as an Alternative to the Rabbit Pyrogen Test for Mono- and Multi-Component Shigella GMMA-Based Vaccines. 2021. 9(7): p. 1375.
  8. Cirefice, G., et al., The future of pyrogenicity testing: Phasing out the rabbit pyrogen test. A meeting report. Biologicals, 2023. 84: p. 101702.
  9. Allen, D., et al. Using the Monocyte Activation Test for medical devices. in NICEATM poster: SOT 2019 Annual Meeting. 2019.
  10. Sandle, T., FDA guidance on pyrogens and endotoxin testing.