Absence of pyrogens – an important quality attribute for injectables and medical devices
Pyrogens are fever-inducing substances of various origins and molecular structures that are recognized by the cells of the innate immune system. Fever is part of the reaction of the innate immune system to invading microbes. Microbial products or so-called pathogen-associated molecular patterns are recognized by Toll-like receptors (TLRs) of phagocytic cells (monocytes/macrophages, neutrophils) and endothelial cells lining the inner surface of blood vessels and capillaries1. TLR-activation leads to secretion of cytokines, predominantly IL-1, IL-6 and TNF (by macrophages) or IL-1 and IL-8 (by neutrophils,2). The cytokines IL-1, IL-6 and TNF act as endogenous pyrogens on the thermoregulatory center of the hypothalamus to induce prostaglandin E2 production and to raise the hypothalamic thermostatic set point1. Depending on the amount of pyrogenic material, the induced response can range from mild increases in body temperature to septic shock.
Detection of pyrogens – past and present
Reliable detection of pyrogenic contaminations is difficult, as pyrogens are a chemically diverse group of molecules only characterized by their effect on the human innate immune system. An ideal detection method would be able to detect any pyrogen able to induce a reaction of the human immune system, irrespective of the nature of the offending molecule.
Traditionally, pharmaceutical products and medical devices are tested for pyrogen content using the rabbit pyrogen test, which monitors the body temperature of rabbits after injection of a preparation of the product to be tested. An alternative method in frequent use is based on the lysate of blood cells of the horseshoe crab. Limulus amoebocyte lysate (LAL) coagulates in the presence of a certain type of pyrogen, a lipopolysaccharide (endotoxin) derived from the cell wall of gram-negative bacteria. Although limited to one type of bacterial pyrogen, this test has been very useful, especially to detect contaminations introduced into drug preparations due to contaminated water systems.
Today, quality control for presence of pyrogens is getting more and more complicated, as the production processes for biotechnology and cell therapy products entail the risk of various contaminants entering the final product, such as viruses from animal-based raw materials or gram-negative and gram-positive bacteria from contaminations (see Fig. 1).
With the recent revision of the general chapter for pyrogen testing in the European Pharmacopoeia (chapter 5.1.10), it is now necessary to evaluate the product, production process and raw materials with respect to the risk for pyrogens that are non-detectable by the bacterial endotoxin test. This proof-of-absence is difficult to obtain without testing for other pyrogens (see Fig. 2), meaning that a universal pyrogen test is needed even when the final quality control assay test is supposed to be the bacterial endotoxin test. This universal test is the monocyte activation test, which essentially monitors the pyrogen detection by TLR-expressing human cells and the subsequent production of cytokines or endogenous pyrogens.
The monocyte activation test - A new robust and easy-to-use assay format
The monocyte activation test has been described as in vitro-alternative to the rabbit pyrogen test and has been included into the European Pharmacopoeia in 2010. The test systems currently available all rely on primary human blood or cells isolated from blood donations. Aside from the concern that human blood is a valuable resource already in high demand by hospitals, primary human material is susceptible to donor-related variations in response, leading to assay systems that are difficult to standardize. Until now, this has hampered the use of the monocyte activation test in quality control.
The obvious solution to this problem would be the use of a pyrogen-responsive cell line for the monocyte activation test. While this was shown to be possible for the Mono Mac 6 cell line already in 2005 3, the culture of monocytes as pyrogen-responsive cells requires solid experience in cell culture and has been difficult to implement in quality control laboratories.
Solvias has now developed a test system based on the human monocytic cell line Mono Mac 6 in a ready-to-use format. The cell line is prepared under standardized conditions, leading to robust test performance independent of the batch of the cells used (see Figure 3). With the development of ready-to-use cells, the test can now be performed in any laboratory equipped with aseptic work benches and plate readers to determine optical densities. Neither specific cell culture experience nor the usual cell culture equipment are necessary. The test performance was evaluated using various known pyrogenic substances (see Fig. 3). As the cells can be stored for long periods in a freezer and only need to be thawed to perform the assay, the new monocyte activation test now combines the advantages of ease-of-use of the primary cells with the robustness in performance of a cell line.
In our laboratories, the monocyte activation test has been successfully applied to a variety of test items, ranging from blood products to vaccines and medical devices. The latter is a particular advantage of the monocyte activation test. Medical devices can be brought into direct contact with the test system, thus allowing detection of surface-bound pyrogens.
The assay is currently available as an analytical service in our laboratories and is available as a MAT kit (the PyroMAT™ System) through MilliporeSigma – the life science business of Merck. An announcement on our collaboration with MilliporeSigma can be found here.
1 Dinarello C, Review: Infection, fever, and exogeneous and endogeneous pyrogens: some concepts have changed, Journal of Endotoxin Research 10 (2004), 201 – 222
2 Hayashi F et al., Toll-like Receptors stimulate human neutrophil function, Blood 102 (2003), 2660-2669
3 Hoffmann S et al: International validation of novel pyrogen tests based on human monocytoid cells, Journal of Immunological Methods 298 (2005) 161-173
Fig. 1 Pyrogens and detection methods
Fig. 2 Choice of pyrogen detection method
Fig. 3 Monocyte activation test based on ready-to-use cells