Before parenteral drugs are released onto the market, they have to be tested for certain toxins known as pyrogens. As fever-inducing substances, they can be life-threatening to patients when introduced into the body through injectable pharmaceuticals. A number of pyrogen detection tests can therefore support companies in making their drugs safer.
Pyrogens are mostly of microbial origin and their molecular structure is greatly diverse. Many are bacterial toxins belonging to either of the two subgroups, endotoxins and exotoxins. While exotoxins are released outside of the cell, endotoxins are contained within the cell. Both exotoxins and endotoxins can activate the human immune system and trigger systemic reactions, such as fever.
The Rabbit Pyrogen Test
Pyrogen detection tests are becoming increasingly important for the analysis of fever-inducing substances in parenteral drugs. Though several such tests exist, the Rabbit Pyrogen Test (RPT) remains commonly used for the detection of pyrogens.
“Considered as the most reliable animal model for pyrogen detection, the rabbit-based test intends to reproduce the fever reaction that occurs after the injection of a contaminated drug,” explains Laure Robert, Global Product Manager at Merck. “If pyrogens are present in a sample, the rabbit will have a rise in temperature indicating contamination.”
However, the RPT is associated with a number of limitations, including low sensitivity, poor robustness, and high consumption of animals. Many companies choose to outsource the Rabbit Pyrogen Test, as they do not own animal housing facilities. This often results in long and tedious processes, paperwork, and high costs.
Furthermore, the test goes against the principle of replacing, reducing and refining (the 3Rs principle) the use of animals in the lab as described in the EU Directive 2010/63/EU for the Protection of Animals Used for Scientific Purposes. These regulations and guidelines emphasize the importance of using in vitro pyrogen detection methods over the Rabbit Pyrogen Test for the sake of animal welfare.
The Limulus Amoebocyte Lysate Test
The pyrogen detection method mostly used for quality control testing is based on the lysate of blood cells from the horseshoe crab. “Limulus amoebocyte lysate, LAL in short, coagulates in the presence of an endotoxin derived from the cell wall of Gram-negative bacteria,” Laure explains.
Although the LAL method is limited to the detection of endotoxins, the test has proved very useful in the past, for example, for the detection of toxins introduced into pharmaceuticals due to contaminated water systems. It is also faster, cheaper, and more sensitive than the RPT.
Nevertheless, companies willing to perform the LAL test rather than the RPT, take the risk of not detecting some of the potential contaminants that result from the production process. Current guidelines for this test therefore state that an additional risk assessment has to be performed to rule out the presence of non-endotoxin pyrogens in the product.
We need better pyrogen detection more than ever before
Never have we needed new toxin detection methods more than today. “The current evolution of production processes, such as biologics and cell therapy production, carries an additional risk of various contaminants entering the final product, including viruses from animal-based raw materials or Gram-negative and Gram-positive bacteria,” Laure explains.
“That’s why current tests for the detection of endotoxins are limiting and not sufficient to ensure patient safety. Consequently, there is a critical need for an in vitro test that can fully replace the rabbit pyrogen test, and detect a broad range of pyrogens, not only endotoxins.”
The Monocyte Activation Test
Currently, the only available replacement for the RPT and for the assessment of non-endotoxin pyrogens is the Monocyte Activation Test (MAT). This in vitro test method can detect all pyrogens that activate the Toll-Like Receptor (TLRs) pathway, including endotoxins and non-endotoxin pyrogens (NEPs).
Based on the actual human immune response against pyrogens, the tested sample is incubated with human monocytes, a type of white blood cell. If pyrogens are present, the monocytes are activated via the TLRs, which results in a release of cytokines, pro-inflammatory molecules. The cytokines are detected with the help of the enzyme-linked immunosorbent assay, known as ELISA. The ELISA technique involves specific antibodies and an enzymatic color reaction.
“Unlike the LAL test, the Monocyte Activation Test is not limited to endotoxin detection but is effective for both endotoxins and non-endotoxin pyrogens,” says Laure. “Although the MAT does not give results as fast as the LAL test, it is currently the only test for the detection of a broad range of pyrogens and compliant with the EU regulation aimed at decreasing animal testing. Giving results within 1.5 days, it remains a convenient solution for pyrogen detection in a quality control lab”
An easy method for pyrogen detection: the PyroMAT™ System
For users, the MAT is highly convenient, as it is now available in ready-to-use kit formats. Merck, for example, has recently marketed a new MAT kit, known as the PyroMAT™ System.
“The PyroMAT™ System uses human monocytic cells from the Mono-Mac-6 cell line as a source of monocytes for the reaction,” Laure explains. “The response to pyrogens is determined by the measurement of Interleukin-6 (IL-6) produced by the monocytes. For this purpose, the ELISA microplate supplied in the kit is coated with an antibody specific to IL-6.”
The monocytic cell line is ready to use, which spares users the need for cell culture equipment and competencies, and enables standardized reactivity and high sensitivity for pyrogen testing. “The PyroMAT™ System also comes with a data analysis tool making statistical analysis and data interpretation easy and more comfortable for the end user,” Laure adds.
In its new ready-to-use kit format, the MAT allows users to easily detect a wide range of pyrogens, including both endotoxins and NEPs. The test is helping companies to ensure product quality and patient safety and adheres to the principle of replacing, reducing and refining the use of animals in labs.
Images via Kateryna Kon, lunamarina, Jarun Ontakra/Shutterstock.com; Laure Robert/LinkedIn; Merck Group
Author: Larissa Warneck, Science Journalist at Labiotech.eu