Pyrogen Detection: Want to stop using animal based methods and get higher sensitivity?

Pyrogen Detection: Want to stop using animal based methods and get higher sensitivity? 

Ready-to-use PyroMAT™ in vitro system 

We are glad to inform you that our new in vitro solution for pyrogen detection is now available: our PyroMAT™ system is the only cell-line based Monocyte Activation Test (MAT) provided as a ready-to-use kit on the market.

The PyroMAT™ system is based on the use of the Mono-Mac-6 cell line: it is designed to provide a standardized reactivity. Once these human monocytes come into contact with pyrogens from a contaminated sample, they produce cytokines such as interleukin-6 (IL-6), which can be detected with enzyme-linked immunosorbent assay (ELISA).

With the PyroMAT™ System, we offer a new robust and sensitive solution for pyrogen detection supported by international regulations and guidelines to reduce animal consumption for pyrogen tests. Make the move to in vitro methods, and benefit from our expertise:

The advantages of monocyte activation test… 

• Detection of the full range of pyrogens to ensure patient safety. Like the rabbit pyrogen test, MAT is effective in the detection of both endotoxins and Non-Endotoxin Pyrogens (NEP). 
• Extended range of testable products: the most frequently applied methods are limited in the product types they are able to test. The MAT offers more flexibility regarding its applications. 
• In vitro assay that mimics the human immune reaction: for a robust predictive model that reduces animal consumption. • Compliance with international regulations and guidelines in line with ethical trends of industry and regulatory authorities to decrease the use of animal based testing. ... Combined with the benefits of using a cell line • Standardized reactivity and high sensitivity (LOD 0.05 EU/mL) 
• Convenience of a ready-to-use cell line to avoid laborious lab work to maintain the cell line and avoids the need of a culture cell laboratory. • Qualified cells: in addition to being cited in the international validation of MAT, MonoMac-6 cells are qualified for the detection of all types of pyrogens via testing of the expression of all surface Toll-Like Receptors (TLRs). 

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Monocyte Activation Test (MAT) - The in vitro test for pyrogen detection – White paper

Monocyte Activation Test (MAT)

The in vitro test for pyrogen detection

Pyrogens…a hot story

Adverse reactions to parenteral preparations have been described as early as the late 19th century, frequently termed “injection fever”. The first fever causing agents, “pyrogens”, were identified in 1912 by Hort and Penfold, who were also the first to design a pyrogen test based on injection of material into rabbits. At that time, the pyrogenic agent was identified as endotoxins included in preparations of gram-negative bacteria. Interestingly, it was shown that live and dead microorganisms presented the same pyrogenic potential.

In the following years, it became more and more clear that sterility is not necessarily equal to apyrogenicity, which led to the inclusion of a pyrogen test in the 12th edition of the United States Pharmacopoeia (USP) in 1942.

Due to their stability, endotoxins can be very difficult to remove by classical bactericidal procedures such as heating or filtration. This made control of the whole production process necessary, especially for the water used, as this water was frequently found as source of pyrogenic contaminations.

The high number of pyrogen tests on rabbits and the variable sensitivity of that test system (e.g. by development of pyrogen tolerance in rabbits after repeated injections) made development of alternative tests necessary. The first and most successful of these new tests was the bacterial endotoxin test based on the lysate of amoebocytes from the blood of horseshoe crabs, which became commercially available in the 1970s and has been widely used as a replacement for the rabbit pyrogen test.

Today's qualified water systems no longer present such a high risk of endotoxin contamination, with more than 99% of our tests for various production sites showing contamination of much less than the specification of 0.25 EU/mL.

On the other hand, quality control for the presence of pyrogens is getting more and more complicated, as production processes (e.g. biotechnology and cell therapy products) bring new risks of various contaminants (i.e Non-Endotoxin Pyrogens) entering the final product, like viruses from animal-based raw materials or gram-negative bacteria from contaminations. Non-Endotoxin Pyrogens (NEPs) are undetectable by the bacterial endotoxin test,

and there is therefore a risk of overlooking a NEP contamination.

In 2016, due to the increase in production of more and more complex products, the general chapter for bacterial endotoxins testing in the European Pharmacopoeia (chapter 5.1.10) introduced the necessity for an evaluation of the product, production process and raw materials with respect to the risk for pyrogens that are non-detectable by the bacterial endotoxin test (ie Non-Endotoxin Pyrogens).

In this context, the in vitro pyrogen test based on human cells offers a valuable alternative to the rabbit pyrogen test. Since2010, the Monocyte Activation Test has been described as a compendial method for Pyrogen Detection in the European Pharmacopeia (chapter 2.6.30).

Test comparison

Both RPT and LAL tests are animal-based methods. LAL cannot can adequately detect the full spectrum of Pyrogens. Moreover, such tests cannot be used on several pharmaceutical products or for the testing of solid materials such as medical devices.

Click here to download our 12-page White paper.

Non-endotoxin Microbial Pyrogens

Pyrogens of microbial origin need to be considered in relation to the risks they pose to pharmaceutical products. Despite the low theoretical risk posed by non-endotoxin pyrogens, questions of their occurrence and risks are sometimes raised by regulators through inspections and by assessors during license changes and submissions. When such questions arise the pharmaceutical manufacturer is often called upon to undertake a risk assessment. Some of the information outlined within this article could be helpful in developing such an assessment.

With this regard, Tim Sandle has written an article for the Journal of GXP Compliance.

The reference is:

Sandle, T. (2015) Assessing Non-endotoxin Microbial Pyrogens in Relation in Pharmaceutical Processing, Journal of GXP Compliance, Vol. 19, Issue 1, 1-10

For details see: IVT.

If you are interested in reviewing this, please contact Tim Sandle.

Posted by Tim Sandle