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.
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