Since July 1, 2025, the European Pharmacopoeia (EP) 11.8 has officially deleted the Rabbit Pyrogen Test (RPT), making the Monocyte Activation Test (MAT) the only recommended alternative method. This transformation is not only a core component of Europe’s overall regulatory strategy to reduce animal testing, but also marks that in vitro detection methods will gradually dominate quality control in the future.

Faced with this mandatory regulatory change, how to efficiently complete the MAT method transition and successfully pass the application has become a core concern for pharmaceutical companies. Combined with the latest requirements of EP 2.6.30, this article shares the practical experience and key application points of Huzhou Shenke Biotechnology Co., Ltd. (HZSKBIO^®) in assisting clients with obtaining CEP approval for active pharmaceutical ingredients (APIs).

01 Regulatory Background: MAT’s Global Regulatory Synergy from "Optional" to "Mandatory"

Since MAT was incorporated into the European Pharmacopoeia (Chapter EP 2.6.30) as an alternative detection method in 2010, it has now become the sole method for evaluating the full spectrum of pyrogens.

· The U.S. Food and Drug Administration (FDA) issued an industry guideline in 2012, recognizing MAT as an alternative pyrogen detection method. The United States Pharmacopeia (USP) has also listed it as an alternative method.

· MAT is officially designated as a pharmacopoeial method in the Russian and Eurasian Pharmacopoeias.

· It is included as an alternative method in the pharmacopoeias of China, India and Brazil, and as a supplementary method in Japan.

This regulatory update in EP 11.8 means that manufacturers of injections, biological products and medical devices marketed or sold in the European Union must focus on the following compliance requirements:

1. Method Transition: Establish and validate MAT detection capabilities as soon as possible, or entrust qualified institutions to perform testing;

2. Document Update: Revise quality control methods and relevant Standard Operating Procedures (SOPs) in registration dossiers accordingly;

3. Compliance Application: Submit the corresponding type of change application in accordance with regulatory guidelines.

02 Core Updates: Key Points for MAT Application Under EP 11.8

In line with the latest regulatory requirements, successful MAT method application for product quality control requires focus on the following critical steps:

Prioritize Risk Assessment

· Conduct a comprehensive pyrogen contamination risk assessment based on product attributes, manufacturing processes and raw material sources;

· If only the Bacterial Endotoxin Test (BET) is adopted, scientific data must be provided to prove the absence of Non-Endotoxin Pyrogens (NEPs) in the product;

· Recommendation: Introduce the MAT method during process development and establish a complete NEP control strategy.

Of particular importance is the NEP detection requirement: As explicitly stipulated in EP 5.1.13 Pyrogenicity, the BET method may be used alone only if the presence of non-endotoxin pyrogenic substances can be scientifically ruled out; if NEPs cannot be excluded, MAT must be used for detection.

In addition, EP 11.8 has made detailed revisions to MAT method standardization, quality control and result acceptance criteria, representing significant changes compared with the previous EP 11.0 version.

03 Typical Case Sharing: Successful Approval Practice

As one of the earliest domestic enterprises to assist clients in replacing the EP rabbit pyrogen test in the European Union, HZSKBIO^® has accumulated extensive experience in MAT regulatory interpretation, method validation and application support.

Recent Practical Achievements

In 2025, HZSKBIO^® assisted clients in completing sample testing, validation and application for the EP 2.6.30 MAT method, and successfully obtained official EU approval in early 2026.

Core Capability Support

(1) Compliant Reagent Assurance

· Compliant cell sources: Authorized for commercial use by ATCC, ensuring clear traceability;

· Authoritative performance validation: Joint performance verification with authoritative institutions, fully compliant with EP 2.6.30 standards;

· Wide applicability: Covers antibodies, vaccines, blood products, traditional Chinese medicine injections, medical devices, chemical drugs, raw and auxiliary materials and other product types.

(2) Professional Technical Support

· One-stop MAT solution: Full-process technical support from method development, validation to application dossier preparation;

· In-depth regulatory interpretation: Real-time tracking of EP, USP, ChP and other global regulatory updates to accurately grasp regulatory trends;

· Proven delivery experience: Reduce trial-and-error costs for clients during method transition based on successful cases.

04 Application Strategy: Key Steps of MAT Validation

Based on successful case experience, the core MAT application process is summarized as follows:

Step 1: Method Selection and Kit Evaluation

As a pharmacopoeial method of the European Pharmacopoeia, MAT does not require full method validation—only sample applicability validation is needed. However, the selected MAT kit supplier must have completed comprehensive performance validation in compliance with ICH Q2(R2), including linearity, range, limit of detection, limit of quantitation, intermediate precision, repeatability, specificity and other indicators.

Step 2: Sample Applicability Validation

Mandatory Checklist

· Qualified endotoxin standard curve (R² > 0.975);

· The test preparation does not affect monocyte IL-6 expression;

· The test preparation does not interfere with IL-6 release detection;

· The test preparation does not interfere with Non-Endotoxin Pyrogen (NEP) recovery.

NEP Validation Requirements

· Validate the detection system with at least 2 NEP ligands;

· Spike at least 1 NEP into the test preparation;

· Positive samples confirmed to be NEP-contaminated in historical batches may be used preferentially.

05 Conclusion: Seize the Regulatory Transition Window

The deletion of the rabbit pyrogen test in EP 2.6.8 marks the European Pharmacopoeia’s full transition from animal testing to in vitro methods in pyrogen detection. Since 2026, EDQM on-site inspections have explicitly listed "absence of an NEP control strategy" as a high-frequency deficiency item. For enterprises yet to complete MAT method transition, the compliance window is rapidly closing.

Leveraging accumulated experience in independently developed reagents, HZSKBIO^® delivers tailored solutions for sample-specific needs and directly addresses "supplementary information" requests in EU/US applications. We support enterprises in completing key technical changes and NEP risk assessment, provide sample pretreatment method development and full validation protocols for diverse sample types, resolve compliance uncertainties at the source, and efficiently accelerate product globalization.

If you are preparing for MAT method validation or facing application-related challenges, please contact us for dedicated technical support and regulatory interpretation materials.