IMPACT: THE MONOCYTE-ACTIVATION TEST (MAT) REDUCES THE RISK OF PYROGENICITY
Fever usually is a useful reaction that helps the individual to overcome attacks from outside the body. Many pathogens share structures (PAMP s, pathogen associated molecular patterns) which allow the innate immune system of multicellular organisms to immediately mobilize a non-antigen- specific defense, whereas the adaptive immune system of vertebrates takes days to mount a specific response against individual antigens encountered for the first time. Recognition of PAMP s triggers a complex cascade of reactions resulting in the release of cytokines (IL-1, IL-6, TNF) which among other effects induces fever (1). This response in case of a local infection is beneficial to activate a massive barrier against the spread of an infective agent in the body. However, a systemic response of the same type bears the risk of harmful reactions including a septic shock.
Therefore it is essential that pharmaceutical products administered intravenously or intrathecally are essentially free of pyrogens. This demand can not be appropriately addressed by sterility tests as many of the pyrogenic structures persist after elimination of living pathogens and bacterial contaminations are not the only source of pyrogenic substances (Fig. 1).
Test procedures and accepted limits of pyrogen contamination are detailed in national pharmacopoeias (2, 3). In the past the two canonical methods were the rabbit pyrogen test and the Limulus Amebocyte Lysate (LAL) test, both with specific advantages and disadvantages. A summary of limitations and disadvantages of these methods is depicted in table 1.
The LAL test shows the best sensitivity to endotoxin but on the other hand is limited to this class of pyrogens originating from gram negative bacteria (4). The rabbit test covers a broader range of substances and is closer to the human immune system but less sensitive and animal consuming. Since April 2010 the Monocyte-Activation Test (MAT ) is included in the European Pharmacopoeia (5) as a third method.
Recommendation
Bernried, Germany18/19 March 2025
Low Endotoxin Recovery/Masking - Hands-on Laboratory Training Course
The MAT combines a short culture for some hours of monocytes from human blood with the potentially pyrogenic material and a test for detection of the induced cytokine.
The attractiveness of this approach derives from the assumption, that the MAT reflects the reactions of the human innate immune system better than the other two methods and thus allows a more realistic prediction of the pyrogenic activity in a probe (Fig. 1).
Fig. 1: Comparison of the course of a pyrogenic reaction in humans with the test procedure of the Monocyte-Activation Test
The potential of the MAT to detect non-endotoxin pyrogens is illustrated by the example of Lipoteichoic acid (LTA ), a pyrogenic cell surface component of gram-positive bacteria (Fig.2).
Fig. 2: Pyrogenic activity of Lipoteichoic Acid expressed in Endotoxin-Units (EU) as detected in the Monocyte Activation Test (MAT) in comparison to the reactivity of the Limulus Amebocyte (LAL) -Test.
As expected, LTA is not detected by the LAL-Test, whereas the MAT clearly detects LTA down to less than 1 μg/ml. In addition the MAT opens the chance to test for pyrogens not only in solution but also on solid materials (6).
Three sources of monocytes are listed in the EP: fresh or cryo-preserved human blood or continuous cell lines. Care has to be taken to eliminate potential variations between blood donors or individual donations if complete human blood is used. In a cell line changes in the repertoire of receptors or other components involved in the induction of cytokine responses would negatively affect the reliability of the test. Whereas the latter risk requires continuous quantitative controls of a multiplicity of functions the first risk can be eliminated by the use of pools from qualified donors. From the practical point of view it appears that aliquoted, cryo-preserved pools of quality controlled donations from several donors are the best source of blood to assure reproducibility of the test results.
Recommendation
Bernried, Germany20/21 March 2025
Monocyte Activation Test (MAT) - Hands-on Laboratory Training Course
The principle proof of feasibility for this combination is documented by a validation reported in 2005 (7, 8). Besides the inclusion of the MAT in the EP the availability of an appropriate test system comprising the culture components as well as the cytokine detection parts is an important step for the utilization of the method in routine work. Time has come to make use of the new chances of improved pyrogen testing in order to increase the safety of products from pharmaceutical industry.
Author:
Dr Bodo Holtkamp
... has been with Biotest AG in Dreieich, Germany, since 1985. Currently he is Senior Director in the GBU Microbiology, heading the department Molecular Biology.
Source:
(1) Takeda, K. and Akira, S. Toll-like receptors in innate immunity, Int. Immunol. 17 (2005), 1-14.
(2) European Pharmacopoeia Chapter 2.6.8. Pyrogens
(3) European Pharmacopoeia Chapter 2.6.14. Bacterial Endotoxins.
(4) Brandenburg, K., Howe, J., Gutsman, T., and Garidel, P., The expression of endotoxic activity in the Limulus test as compared to cytokine production in immune cells. Curr Med Chem. 16 (2009), 2653-60.
(5) European Pharmacopoeia Chapter 2.6.30 Monocyte-Activation Test
(6) Mazzotti, F., Beuttler, J., Zeller, R., Fink, U., Schindler, S., Wendel, A., Hartung, T., and von Aulock, S., In vitro pyrogen test - A new test method for solid medical devices. J. Biomed. Mater. Res. 80 (2007), 276-282.
(7) Hoffmann, S., Peterbauer, A., Schindlerm S., Fennrich, S., Poole, S., Mistry, Y., Montag-Lessing, T., Spreitzer, I., Löschner, B., van Aalderen, M., Bos, R., Gommer, M., Nibbeling, R., Werner-Felmayer, G., Loitzl, P., Jungi, T., Brcic, M., Brügger, P., Frey, E., Bowe, G., Casado, J., Coecke, S., de Lange, J., Mogster, B., Naess, L. M., Aaberge, I. S., Wendel, A., and Hartung, T., International validation of novel pyrogen tests based on human monocytoid cells, J. Immunol. Meth. 298 (2005), 161-173.
(8) Schindler, S., Spreitzer, I., Löschner, B., Hoffmann, S., Hennes, K., Halder, M., Brügger, P., Frey, E., Hartung, and T., Montag, T., International validation of pyrogen tests based on cryopreserved human primary blood cells, J. Immunol. Meth. 316 (2006), 42-51.
