Preface

• Starting from July 1, 2025, the supplement 11.8 of the European Pharmacopoeia eliminates the rabbit pyrogen test and recommends the Monocyte Activation Test (MAT) as a suitable alternative.

• From July 20, 2025, the Testing Method for Bacterial Endotoxins in Medical Devices – Recombinant Factor C Method (YY/T1939-2024) will be officially implemented.

Pyrogen testing and endotoxin testing are crucial quality control procedures in the production of biologics, cell products, injectable drugs, and medical devices. Pyrogen testing targets all fever-inducing substances, while endotoxin testing detects only endotoxins and cannot identify non-endotoxin pyrogens.

Relationship Between Pyrogens and Endotoxins

Pyrogens

Substances that induce a fever response when injected into warm-blooded animals. These include bacterial pyrogens (e.g., endotoxins), endogenous pyrogens (e.g., interferons, TNF, IL-6), exogenous pyrogens (e.g., bacteria, viruses, fungi, spirochetes, plasmodium), and chemical pyrogens (e.g., phthalates).

Characteristics: Heat-resistant, filterable, water-soluble, and non-volatile. Symptoms like high fever and sweating typically appear about 0.5 hours after entry into the human body, with fever reaching up to 40°C.

Detection Methods: Rabbit Pyrogen Test (RPT) (ChP/USP), Monocyte Activation Test (MAT) (ChP/USP/EP), Reporter Gene Assay (ChP).

Endotoxins

Toxins released from the cell walls of Gram-negative bacteria (e.g., Salmonella, Shigella), considered a type of pyrogen. Composed primarily of lipopolysaccharides (LPS), including an O-specific polysaccharide, a core polysaccharide, and lipid A.

Characteristics: Harmless when ingested, but can trigger severe reactions when introduced into the bloodstream via injection. Even trace amounts can cause a fever, which usually resolves after about 4 hours.

Detection Methods: LAL assay (ChP/USP/EP), Recombinant Factor C method (ChP/USP/EP)

Regulatory Updates

Pyrogen and endotoxin tests are mandatory in all major pharmacopoeias. Traditional detection methods rely on animals. However, with advancing technologies and growing emphasis on animal welfare, detection methods that align with the 3R principles (Reduction, Refinement, Replacement) have gained regulatory priority.

Endotoxin Removal Strategies

Common sources of endotoxins in biologics include: microbial strains, production environment, non-standard operations, equipment and devices, and raw materials. Moist heat sterilization (121°C for 1 hour) cannot destroy endotoxins. Effective methods include dry heat sterilization (180°C for 3–4 hours, 200°C for 60 min, or 250°C for 30–45 min), or treatment with strong acids or alkalis.

Solution 01
Newly Upgraded, Highly Interference-Resistant Recombinant Factor C Endotoxin Detection Kit

During endotoxin testing, complex samples often contain various metabolic by-products, high concentrations of nutrients, formulation components, residual denaturants and chelators, ions, and surfactants, which can severely interfere with the results.

These substances may inhibit the active components of the test reagent or bind endotoxins into tightly wrapped complexes that mask the active sites, preventing proper detection and leading to inaccurate readings.
The conventional solution is sample dilution, but when the necessary dilution exceeds the Maximum Valid Dilution (MVD), recovery remains unacceptable, and results are unstable.

Dispersant 1

Dispersant 2

The Hzymes Recombinant Factor C Endotoxin Detection Kit has been fully upgraded!

It now includes a range of interference-resistant dispersants tailored for different sample types, enabling accurate endotoxin detection in complex matrices.

Greatly Expanded Sample Compatibility

With the upgraded dispersants, the kit can now handle a broader range of challenging samples: abnormal pH, lack of divalent cations, high osmolarity, fermentation supernatants, culture media, plasmids, mRNA, aluminum adjuvants, etc.

High Precision Detection Results

The new dispersants minimize the impact of interfering substances on the reaction system, ensuring more accurate test results.

Improved Repeatability and Stability

By reducing result variation caused by fluctuating levels of interfering substances, the upgraded dispersants significantly enhance test repeatability and consistency.

Solution 02
Automated Data-Processing Microplate Reader

The Recombinant Factor C method relies on a fluorescence-based reaction (Ex = 380 nm, Em = 440 nm) detected using a microplate reader. Conventional readers often feature complex user interfaces, require manual data processing, and are costly.

Our multi-functional plate reader offers a streamlined interface, simple operation, and automated data analysis—greatly improving lab efficiency.

Access Control

Four-tier user access with separate accounts and passwords.

Intuitive Display

Clearly laid out functional modes and simple parameter settings. Programs and data are saved independently for easy retrieval.

Automated Data Analysis

Offers multiple data processing modes, including blank subtraction, standard curve generation, and qualitative analysis.

Remote Data Transfer

Instrument data can be uploaded to a server for real-time remote access and review.

Solution 03
Pyrogen Detection Kit

Although the traditional rabbit test remains the “gold standard” for pyrogen testing, it has notable limitations:

• Low Sensitivity: Risk of false negatives
• Cumbersome Operation: Requires specialized animal facilities and procedures
• High Biological Variability and Animal Dependency

The new MAT method enables in vitro detection of non-endotoxin pyrogens such as Gram-positive bacteria, fungi, and viruses. As the only method accepted by the European Pharmacopoeia, switching to MAT in line with evolving international regulations is essential for successful product registration.

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