Revolutionizing Pyrogen Detection: Minerva Biolabs Launches the NAT-MAT® System

In the pharmaceutical industry, pyrogen detection is mandatory to avoid life-threatening fever reactions that can be induced by both microbial and non-microbial substances. Traditionally, this test relied on animal-based methods, like the rabbit pyrogen test (RPT). With increasing awareness of ethical concerns and growing demand for alternatives, the Monocyte Activation Test (MAT) was introduced into the European Pharmacopeia (EP) in 2010 – providing a human in vitro system to detect and quantify pyrogenic substances.

In June 2024 the Ph. Eur. Commission has decided to end the RPT era by deleting the RPT from the Ph. Eur.. The revised texts omitting the RPT and the new general chapter on Pyrogenicity (5.1.13) will be implemented in Supplement 11.8 of the Ph. Eur. on 1 July 2025 ^[1]. For this reasons, regulatory authorities currently encourage manufacturers to implement MAT in their QC system. Other pharmacopeias, such as the United States Pharmacopeia, recognize the MAT as an alternative method to RPT.

What is the NAT-MAT® and How Does it Work?

The MAT is based on the activation of monocytes by pyrogenic substances present in the sample. Upon exposure to potential pyrogens, monocytes undergo a signalling pathway resulting in secretion of pro-inflammatory cytokines, such as IL-1β, TNFα and IL-6, that play an important role in the fever reaction. The release of these cytokines is measured in the MAT.

In contrast to conventional MAT kits, that measure the protein secretion of cytokines using ELISA, the NAT-MAT® measures the gene expression of IL-1β and TNFα using digital PCR. This enables a shorter treatment time for the cells, resulting in a fast turnaround-time.

Since both endotoxin and non-endotoxin pyrogens can be detected, the NAT-MAT® represents a complete pyrogen test that can replace the RPT. The NAT-MAT® can be conducted as in-process control and final release testing according to Ph. Eur. 2.6.30.

The NAT-MAT® relies on the use of NAT-MAT® Cells which are stimulated with a pyrogen standard and the sample to be tested followed by nucleic acid extraction and subsequent dPCR analysis of the gene expression of IL-1β and TNF-α.

What Components does the NAT-MAT® System Consist of?

NAT-MAT® Cells are cryopreserved ready-to-use HL60-derived macrophages that can be seeded directly into a 96-well plate.

NAT-MAT® Extraction is a magnetic bead-based extraction kit for automated extraction of nucleic acids (e.g. with KingFisher™ Flex).

NAT-MAT® dPCR provides a lyophilized primer/probe mix amplifying two cytokines (IL-1β, TNF-α) and a housekeeping gene in a triplex PCR. IL-1β is detected in the HEX™-channel, TNF-α is detected in the Cy5®-channel and the housekeeping gene is detected in the FAM™-channel.

Prefer an all-in-one solution?

NAT-MAT® All-in-One System offers all necessary components, including NAT-MAT® Cells, NAT-MAT® Extraction and NAT-MAT® dPCR.

Reactivity to Endotoxin and Non-Endotoxin Pyrogens

Certain endotoxin and non-endotoxin pyrogen standards have been tested with the NAT-MAT® system, showing excellent linearity. The assay is able to detect pyrogens that activate Toll Like Receptors 1-9 (extracellular and intracellular receptors).

Endotoxin pyrogens

 

Non-endotoxin pyrogens

  

  

  

Why it Matters – the Top Advantages

What Makes the NAT-MAT® More Accurate?

A special feature of the NAT-MAT® is the normalization of the pyrogen concentration to the cell number, that might vary from well to well. This is possible by parallelly measuring the gene expression of the cytokines and the gene expression of a housekeeping gene. Normalizing the pyrogenic load to the cell number leads to highly accurate and precise results.

What Makes the NAT-MAT® Faster?

ELISA-based MATs measure the protein expression of the cytokine and require overnight stimulation of the cells. In contrast, the NAT-MAT® is based on measuring the gene expression of the cytokine. For this reason, the cells only need to be stimulated for 4 hours.

What Makes the NAT-MAT® More Reliable?

Conventional MAT kits often use PBMCs as a cell source, which can vary widely from lot to lot, leading to inconsistent results. In addition, cell supply with PBMCs can be a challenge.

The NAT-MAT® uses a stable cell line of HL-60 derived macrophages, which ensures reproducible outcomes and a reliable cell supply.

What Makes the NAT-MAT® Save Your Time?

The NAT-MAT® can be highly automated. The extraction process and pipetting can be carried out automatically by robots which saves time and reduces manual steps to a minimum.

What Else Makes the NAT-MAT® Even More Special?

Existing MATs are based on the measurement of only one cytokine. The NAT-MAT® measures the gene expression of two cytokines – IL-1β and TNFα. This enables sensitive and robust quantification across varying pyrogen levels.

We provide support for product specific validation. Please contact support@minerva-biolabs.com.

^[1] https://www.edqm.eu/en/-/ph.-eur.-bids-adieu-to-rabbit-pyrogen-test-in-its-monographs