Monocyte Activation Test Research Articles

Safety testing of cell-based medicinal products: Opportunities for the monocyte activation test for pyrogens

The European Partnership for Alternative Approaches to Animal Testing (EPAA) pointed out the need to involve authorities throughout the process of validation and legal acceptance of alternatives to animal experiments. The Paul-Ehrlich-Institute (PEI), Federal Agency for Sera and Vaccines, is the national competent authority in Germany which is responsible for the quality and safety of biologicals including blood and cell-based products. This paper is intended to contribute to the discussion concerning the use of alternative methods in safety testing of medicinal products and considers the scientific work of the PEI in this field. From a regulator's perspective, adequate demonstration of safety and quality of medicinal products are of major interest. Additionally, the availability of the products to the patient has to be taken into consideration. It has to be carefully explored whether the respective in vitro method for demonstration of non-clinical safety as part of the non-clinical development programme is able to guarantee safety level comparable to the corresponding experiment in animals. The topics cited above shall be discussed in this paper using the example of the Alternative Pyrogen Test or also called Monocyte Activation Test. The Alternative Pyrogen Test could serve as paradigm to exemplify how an alternative test can provide at least a comparable level of safety estimation in comparison with a conventional animal test. Furthermore, this alternative test creates additional information which cannot be obtained from the animal experiment, and might also open further scientific insight into the mechanisms of pyrogenicity and acute pro-inflammatory reactions in patients. This test method allows the definition of pyrogen limits for medicinal products. Due to its use of relevant cell systems this in vitro test might contribute significantly to safety assessments of advanced medicinal products during the pre-clinical phase.

Monocyte activation test for pro-inflammatory and pyrogenic contaminants of parenteral drugs: test design and data analysis

An optimised test designed for an in vitro monocyte activation test for pro-inflammatory and pyrogenic contaminants of parenteral drugs is described, together with ways to address the inherent variability of such assays in which cells are cultured using 96-well plates. The test preparation is cultured with peripheral blood mononuclear cells (PBMNC) and the contaminants in the test article stimulate the release from the cells of the endogenous pyrogenic cytokine interleukin-6 (IL-6). The test system is in use within the pharmaceutical industry and at a national control authority for detecting pro-inflammatory and pyrogenic contaminants, including ‘rabbit-negative’ and ‘LAL-negative’ non-endotoxin pyrogens. Products tested include small molecules, biologicals and vaccines. The PBMNC/IL-6 monocyte activation test has been approved by the US FDA as an ‘end-product release test’ and also is being used for in-process testing.

A rapid ‘one-plate’ in vitro test for pyrogens

A rapid, ‘one-plate’ monocyte-activation test is described for detecting endotoxin and non-endotoxin pyrogens in parenteral medicinal products. The one-plate test offers useful gains over conventional ‘two-plate’ (cell culture plate+ELISA plate) tests in terms of its limit of detection, robustness, speed and cost. The ‘one-plate’ test is likely to be applicable to a wide range of products because it allows less time for product interference in the test. The ‘one-plate’ test utilises pyrogen-free anti-cytokine (interleukin (IL)-6 or tumour necrosis factor α (TNFα)) antibodies (Ab), coated and stabilised onto (pyrogen-free) 96-well plates. Monocytes/monocytic cells, endotoxin (lipopolysaccharides, LPS) standard or sample and (pyrogen-free) second (labelled) Ab are cultured together (usually for 2–4 h) on the Ab-coated plate and then the plate is washed and the ELISA completed. There is no transfer from one plate to another and no (further) incubations of (released) cytokine with, first, coating Ab and, then, developing Ab since these steps have already taken place during the initial cell culture. The rapid, ‘one-plate’ test is readily automated. The preferred readout is IL-6, which gives a limit of detection of 0.015 endotoxin units (EU)/ml with peripheral blood mononuclear cell (PBMNC), 0.03 EU/ml with diluted whole blood and 0.05 EU/ml with a monocytic cell line (MONO MAC 6).

Intracardiac route for epinephrine

Lipoteichoic acid (LTA) is a non-endotoxin pyrogen of a great importance in the pathogenesis of sepsis. The Rabbit Pyrogen Test (RPT) is able to detect all types of pyrogens but involves the use of animals. The Bacterial Endotoxin Test (BET) cannot fully replace the RPT because it only detects endotoxins. The Monocyte Activation Test (MAT) is sensitive to all types of pyrogens and it is based on the same biological mechanism that is responsible for the fever reaction in humans. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) has recommended its use for other pyrogens than endotoxin because its equivalence to RPT can be demonstrated. The aim of this study was to evaluate the pyrogenic responses of the RPT and MAT that was induced by LTA. Different LTA concentrations were assayed by the MAT in parallel to the RPT. The results showed that the MAT was more sensitive than the RPT, demonstrating that the MAT detected LTA. This result may contribute to the acceptance of this test by the Brazilian regulatory agencies as a replacement for the animals used in the RPT.