Monocyte Activation Test Solutions

In vitro pyrogen testing for parenteral drugs and medical devices

Cellular Technology Limited’s Monocyte Activation Test offers a comprehensive alternative to the antiquated Rabbit Pyrogen Test and the Limulus Amebocyte Lysate assay.

With the introduction of the Monocyte Activation Test chapter 2.6.30 in the European Pharmacopeia, CTL-MAT combines CTL’s background in immune monitoring to support the global adaption of the Monocyte Activation Test.

Cellular Technology Limited is the pioneer and industry leader in the development, use and manufacturing of standardized tools for cellular immune assays. CTL’s founding scientists were the first to introduce protocols for functionality loss-free cryopreservation of PBMC in ELISPOT. For over 25 years, CTL has been a leading provider of cryopreserved PBMC and cytokine detection kits.

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Monocyte Activation Test Kits

The CTL-MAT kit enables European Pharmacopoeia compliant in-vitro pyrogen testing. All necessary components are included: Cryopreserved pooled PBMC, IL-6 ELISA, reference standards, reagents, and protocols. All components of the test system are fine-tuned for optimal performance in MAT and are certified pyrogen-free.

Reactivity and sensitivity of the CTL-MAT kit: Typical LPS dose response curve is shown after testing with CTL-MAT Kit. LPS was titrated in the concentration range specified on the X axis. The concentration of IL-6 detected in the culture supernatant after 20h incubation of the PBMC with LPS is shown on the Y axis. Note the high reactivity and sensitivity.

Product	Catalog Number
4-Donor MAT Kit – With Endotoxin positive control	CK450
4-Donor MAT Kit – With Endotoxin and three Non-Endotoxin pyrogen positive controls	CK453
8-Donor MAT Kit – With Endotoxin positive control	CK850
8-Donor MAT Kit – With Endotoxin and three Non-Endotoxin pyrogen positive controls	CK853

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Pooled PBMC

The indicator PBMC have been frozen according to Ph. Eur. 2.6.30 according to proprietary protocols that assure that the cryopreserved cells maintain their full functionality after freeze thawing (see representative data below). CTL-MAT offers PBMC suited for MAT testing also independent of its kits, from single donors or pools of 4 or 8 donors.

European Pharmacopeia 2.6.30 specifies requirements for PBMC to be used in MAT. The PBMC provided in CTL-MAT Kits fulfill the mandated criteria:

PBMC isolation from the Buffy coat of four healthy donors
The blood donors fulfill the quality criteria according to EP 2.6.30, section 5-3
Equal number of cells pooled
Cryopreserved within 4 hours from the blood donation

LPS standard curve with the frozen pooled PBMC after thawing determines the acceptance criteria for the standard curve described in EP. Intra-batch reproducibility of CTL-MAT, four donors pooled PBMC. Four aliquots of PBMC (Vials 1-4, specified by the symbols) from the same lot were tested in the MAT. LPS was titrated in the concentration range specified on the X axis. The concentration of IL-6 detected in the culture supernatant after 20h incubation of the PBMC with LPS is shown on the Y axis. Note the overlapping curves, showing high intra-batch reproducibility of results obtained with CTL MAT kits.

Product	Catalog Number
4-donor European Pharmacopeia 2.6.30 compliant cryopreserved pooled PBMC	C4PP5
8-donor European Pharmacopeia 2.6.30 compliant cryopreserved pooled PBMC	C4PP8

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Services

At CTL, we offer comprehensive Pyrogen Testing services, ensuring your parenteral pharmaceutical products and medical devices meet stringent safety standards. Pyrogens, substances that cause fever when introduced into the body, must be detected to ensure regulatory compliance.

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Principles of the MAT Assay

Pyrogen testing: The detection of pyrogens within parenteral pharmaceutical products and medical devices is a globally mandated safety requirement for manufacturers. Because of considerable shifts in the European Pharmacopoeia (Ph.Eur. 5.1.10) as well as noted limitations of the RPT (rabbit) and LAL (horseshoe crab) methods, MAT testing, in accordance with Ph. Eur. 2.6.30 and 2.6.40 has emerged as a significant in vitro test to evaluate the existence of pyrogens in such products. Learn more

The Monocyte Activation Test: The fever reaction results from monocyte activation through microbial products. The Monocyte Activation Test (MAT) detects this reaction in vitro. Briefly, when performing MAT, PBMC (that have been cryopreserved after their isolation from the human body) are exposed in tissue culture to the test substance. If the test substance contains microbial contaminants, these will elicit IL-6 production in the monocytes, which can be detected by an IL-6 ELISA in the culture supernatant. Learn more

The Monocyte Activation Test (MAT) vs. the Rabbit Pyrogen Test (RPT): The fever reaction elicited in rabbits (and humans) results from monocytes, a subpopulation of white blood cells, being activated in the body through exposure to microbial products, resulting in these monocytes releasing endogenous cytokines, such as IL-6. IL-6 then stimulates the thermocenter of the brain to raise the body temperature, resulting in fever. Learn more

The Monocyte Activation Test (MAT) vs. Limulus Amebocyte Lysate (LAL) and Recombinant Factor C Test: White blood cells of the North American horseshoe crab, Limulus, contain a protein called Factor C, that agglutinates in the presence of lipopolysaccharides (LPS). Relying on this reaction, the Limulus Amebocyte Lysate test (LAL) has emerged as an in vitro alternative for the RPT to detect contaminations with LPS. Unfortunately, at least one third of the crabs do not survive the bleeding. Learn more

Resources

Shabnam Solati: An improved monocyte activation test using cryopreserved pooled human mononuclear cells PDF download

Solati et al. 2022: The Monocyte Activation Test detects potentiated cytokine release resulting from the synergistic PDF download

CTL-MAT medical device poster PDF download

COA – 4-Donor Pooled PBMC (5 Mill.) certificate of analysis (C4PP5-COA-MAT231019P4) PDF download

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The Monocyte Activation Test (MAT) vs. the Rabbit Pyrogen Test (RPT)

People throughout the world should be able to count on parenteral drugs, cosmetics, and biopharmaceuticals to be safe. The same holds true for medical devices. Microbial contamination, however, can happen even under the most stringent manufacturing standards.

Such contaminations can lead to conditions ranging from mild fever to life-threatening septic shock in recipients. This is why regulatory agencies throughout the world mandate "pyrogenicity testing" of every production batch of parenteral drugs and medical devices.

A product's pyrogenicity due to contamination has traditionally been tested by injecting it into rabbits, in the Rabbit Pyrogen Test (RPT). While the RPT is ideally suited to detect microbial contaminants of all kinds, it involves animal testing.

Pyrogens Detected by RPT, MAT, LAL, and rFC

Pyrogen	RPT	MAT	LAL	rFC
Gram-negative bacteria	Yes	Yes	Yes	Yes
Gram-positive bacteria	Yes	Yes	No	No
Fungi, Mold	Yes	Yes	No	No

The fever reaction elicited in rabbits (and humans) results from monocytes, a subpopulation of white blood cells, being activated in the body through exposure to microbial products, resulting in these monocytes releasing endogenous cytokines, such as IL-6. IL-6 then stimulates the thermocenter of the brain to raise the body temperature, resulting in fever.

As the mechanism of this fever reaction is now well understood, it has become possible to model it in vitro via the Monocyte Activation Test (MAT), which is a superior detection system for microbial contaminations compared to the RPT, without involving animal sacrifice. The European Pharmacopoeia has mandated avoiding the RPT whenever possible in favor of in vitro laboratory testing.

MAT has additional advantages over RPT: it's applicable to a greater variety of products and is more precise, reliable, and cost-effective compared to RPT.

CTL-MAT's Test kit and customer support structure have been established to help realize the goal of maximal drug safety while avoiding unnecessary animal cruelty.

MAT vs. LAL (and recombinant Factor C) Test

White blood cells of the North American horseshoe crab, Limulus, contain a protein called Factor C, which agglutinates in the presence of lipopolysaccharides (LPS). The Limulus Amebocyte Lysate test (LAL) has emerged as an in vitro alternative for the RPT to detect contaminations with LPS. Unfortunately, at least one third of the crabs do not survive the bleeding. To avoid unnecessary animal cruelty, the trend is to obtain Factor C not by bleeding these animals but by producing it as a recombinant protein. Irrespective of Factor C's origin, the test principle remains the same, resulting in highly sensitive and specific tests for LPS detection.

Pyrogens Detected by RPT, MAT, LAL, and rFC

Pyrogen	RPT	MAT	LAL	rFC
Gram-negative bacteria	Yes	Yes	Yes	Yes
Gram-positive bacteria	Yes	Yes	No	No
Fungi, Mold	Yes	Yes	No	No

MAT detects all the above microbial contaminants. Monocytes rely on a family of pattern recognition receptors called toll-like receptors (TLR) to identify such targets and eliminate them by phagocytosis. LPS engages TLR-4 on monocytes, triggering them to produce IL-6. By expressing different types of TLRs, monocytes are well-equipped to recognize most microbial agents.

Unlike the LAL test, MAT is uniquely suited to detect microbial contaminations of all kinds beyond LPS. Both LAL and the rFC are compendial methods and need to undergo product-specific validation. The European Pharmacopoeia requires MAT to be used instead of the RPT for the validation of LAL and rFC.