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Improved detection of pyrogens using the monocyte activation test.

* Audrey Borel, PharmD Global Product Manager--Rapid Release Testing Pharma, EMD Millipore

* Bodo Holtkamp, Biotest AG, GBU Mikrobiologie

* Gabriele Schmitz, Biotest AG, GBU Mikrobiologie

* Thomas Hartung, Johns Hopkins University, Center for Alternatives to Animal Testing

Pyrogens are a heterogeneous group of contaminants comprised of microbial and non-microbial substances. The most widely known pyrogen is the endotoxin lipopolysaccharide (LPS), which is produced by gram-negative bacteria. Other microbial substances include those derived from gram-positive bacteria such as lipoteichoic acid (LTA) and particles from yeasts and viruses.

When present in pharmaceutical products, pyrogenic substances can prove harmful to humans by causing life threatening fevers. Therefore, testing of pharmaceutical products for pyrogens is critical to ensure patient safety.

The human response to pyrogenic substances is complex. It is regulated by monocytes, which contain receptors for the various pyrogenic substances and respond with a high sensitivity, especially to endotoxins. The activation starts with LPS binding to toll-like receptor 4 (TLR4) and involves a cascade of intracellular reactions resulting in a release of pro-inflammatory cytokines. The participation of the different receptors in the reaction path is illustrated in Figure 1. There are additional receptors (e.g. TLR2, TLR7, TLR8, TLR9) which, upon binding of their specific ligands, trigger or influence pyrogenic reactions [1].

The traditional methods for pyrogen detection either require animal sacrifice for the Rabbit Pyrogen Test (RPT), or are limited to the detection of endotoxin LPS from the cell walls of gram-negative bacteria (for example in the Limulus Amoebocyte Lysat [LAL] test).

When performing pyrogen testing in pharmaceutical products, the complexity of the human response must be considered. This article will describe a pyrogen detection method based on the Monocyte Activation Test (MAT), which incorporates an assay based on human monocytes conducted in vitro, instead of using animals. Detection of various pyrogenic substances using the MAT and standard methods will be presented.


For analysis of the pyrogenic reactions, the PyroDetect System (EMD Millipore, Darmstadt, Germany), which is based on MAT, was used to assess pyrogens known to interact with different receptors on and in monocytes.

The PyroDetect System uses cryoblood, which is pooled and frozen human whole blood, as a source of monocytes. The monocytes activated by pyrogens produce cytokines which are detected in an enzyme-linked immunosorbent assay (ELISA).

The MAT employing fresh or cryo-preserved human blood was described and validated as an in vitro pyrogen test [2,3]. It has been included in the European Pharmacopoeia (EP) since April 2010 [4].

For cryoblood incubation, sample or endotoxin are mixed with cryoblood in a cell culture plate and kept in an incubator at 37[degrees]C for 8-24 hours for interleukin-1B (IL-1B) production.

For the IL-1B detection, the cryoblood incubation mixture is transferred into an ELISA plate coated with a monoclonal antibody specific to IL-1B. Interleukin molecules present in the culture supernatant are bound by the immobilized antibody. After removal of unbound components by several washing steps, an enzyme-linked polyclonal antibody specific for IL-1B is added. With the addition of the substrate, a color reaction is started, which allows the detection of the bound IL-1B in an ELISA reader.

The pyrogen concentration in the sample is then determined from the IL-1B concentration via an endotoxin standard curve, and analyzed with the PyroDetect data analysis tool.


The broad pyrogen spectrum detected by the PyroDetect system was evaluated against a set of substances.

Lipopolysaccharide is detected by the system down to a concentration of 25 pg/ml, corresponding to 0.25 endotoxin units (EU). This reaction is enhanced by soluble peptidoglycan (PGN) up to a concentration of 10 pg/ml, resulting in a reduction of the limit of detection for LPS by a factor of 10. In addition, a general increase in IL-1B production was observed for all LPS concentrations.

Although this enhancing effect could increase sensitivity, it may create a problem for pyrogen analyses according to the European Pharmacopoeia. If the values for recovery of a defined amount of control endotoxin (spike) exceed the limits of 50 to 200 percent, the test is not regarded as valid [4]. The interference may be overcome by dilution, but is restricted by the contaminant limit concentration (CLC; for intravenous infusions 5 EU/kg body weight/hour) and the limit of detection (LOD) of the test system.

The MAT therefore may not be applicable for substances administered in large volumes. Alternatively, the interfering effect offers the chance for a better differentiation of the inflammatory reactions. In the example of peptidoglycan, the pure substance alone does not induce any 1L-1B response at concentrations between 10 pg/ml and 100 [micro]g/ml (data not shown).

The pyrogenic activity of lipoteichoic acid could be detected down to a concentration of less than 1 [micro]g/ml. The addition of peptidoglycan resulted in similar effects as observed in the reaction to LPS, i.e. a marked reduction of the LOD.

Pyrogenic activation by RNA reported for human TLR8 [5] was confirmed for E. coli RNA as well as synthetic GU-rich 20-mer RNA molecules. As expected, no activation was induced by its counterpart with all uridin residues replaced by adenosin. For E. coli RNA, an LOD of 12.5 [micro]g/ml was calculated; the respective value for the synthetic oligonucleotide was about a factor of 10 lower. The MAT also detects the pyrogenic effect of poly (dT) and its enhancement by the imidazoquinoline compound R848, reflecting the cooperation between the receptors TLR7 and TLR8 [6].


Results demonstrate that MAT is suitable for the detection of various pyrogenic substances which have a direct or indirect influence on fever response in humans. In comparison to the RPT, the sensitivity of the MAT is equal or better [7] but the MAT allows improved standardization without requiring animal sacrifice.

The advantage of the MAT is even more pronounced in comparison to the LAL test which only identifies endotoxins and is subject to false positive reactions to glucans [8].

Detection of the broad spectrum of pyrogens in pharmaceutical products using the MAT allows improved patient safety and offers the chance to abandon animal tests for pyrogen control.

The data presented in this article was originally published in BlOspektrum (2010; 16, 779-781).

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(1.) Takeda K and Akira S (2005) Toll-like receptors in innate immunity. Int Immunol 17: 1-14

(2.) Hoffmann S, Peterbauer A, Schindler S et al. (2005) International validation of novel pyrogen tests based on human monocytoid cells. J Immunol Meth 298: 161-173

(3.) Schindler S, Spreitzer I, Loschner B. et al. (2006) International validation of pyrogen tests based on cryopreserved human primary blood cells. J Immunol Meth316: 42-51

(4.) European Pharmacopoeia 6th Edition (6.7) (2010) Chapter 2.6.30 Monocyte Activation Test

(5.) Heil F, Hemmi H, Hochrein H. et al. (2004) Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303:1526-9

(6.) Jurk M, Kritzler A, Schulte B et al. (2006) Modulating responsiveness of human TLR7and 8 to small molecule ligands with T-rich phosphorothiateoligodeoxynucleotides. Eur J Immunol 36:1815-26

(7.) Poole S and Mussett MV (1989) The International Standard for Endotoxin: evaluation in an international collaborative study. J Biol Stand 17:161-171

(8.) Cooper JF, Weary ME, Jordan FT (1997) The impact of nonendotoxin LAL reactive materials on Limulus amebocyte lysate analyses. PDA J Pharm SciTechnol 51:2-6
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Publication:Pharmaceutical Processing
Date:May 1, 2015
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