EP regulations towards MAT
Internationally, pyrogen testing is enforced as a mandatory step in the process of releasing a batch of any parenteral drug. While the Rabbit Pyrogen Test (RPT) has until now been employed as the default assay, changes in regulation – specifically in the European Pharmacopoeia – has meant the introduction of the Monocyte Activation Test (MAT) as the replacement. In three parts, this page provides a concise but comprehensive overview of the legal underpinnings regarding pyrogen testing, MAT and the suitability of its different possible cell sources from a European Pharmacopoeial perspective.
Transitioning from the Rabbit Pyrogen Test (RPT)
Pharmaceutical producers worldwide are transitioning from the use of the Rabbit Pyrogen Test (RPT) for batch release of their parenteral drugs – as clearly mandated by the European Pharmacopoeia (2.6.8.) in 2017 below.
EP 2.6.8.
"In accordance with the provisions of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, tests must be carried out in such a way as to use the minimum number of animals and to cause the least pain, suffering, distress or lasting harm. Wherever possible and after product specific validation, the [rabbit] pyrogen test is replaced by the monocyte-activation test."
As of the end of 2019, this is now starting to be enforced for all producers wishing to distribute across Europe. Beyond this, RPT has significant shortcomings – specifically its non-specificity towards humans and the fact it solely provides black and white, non quantifiable results.
Employing MAT and/or BET?
In a bid to move away from the use of the RPT, pharmaceutical producers have started to employ Bacterial Endotoxin Tests (BET) like the Limulus Amebocyte Lysate (LAL) or the recombinant Factor C (rFC) assay. Though undoubtedly highly sensitive in detecting the more common endotoxin pyrogens, unlike the MAT, these tests do not also detect non-endotoxin contaminants. As a result, the EP (5.1.10.) stipulates:
EP 5.1.10.
"...the conclusion is generally justified that the absence of bacterial endotoxins in a substance or product implies the absence of pyrogenic components, provided the presence of non-endotoxin pyrogenic substances can be ruled out. The monocyte-activation test (2.6.30) is a suitable method to use to rule out the presence of non-endotoxin pyrogens in substances or products."
Specifically, ruling out the presence of NEP substances requires what the EP identifies as a “risk assessment” (see 5.1.10., section 3) that occurs ‘in process’ or during batch release of any parenteral drug.
However, because the EP's enforcement of this monograph has only just begun for larger manufacturers, routine risk assessments were rarely employed when using BET, and where they were, RPT was used.
The EP's requirement for producers to leave RPT, plus the absence of any alternative in vitro endotoxin pyrogen & NEP test is the basis on which it expressly recommends the use of MAT as a risk assessment where BET is used for batch release.
Ensuring legal and commercial feasibility of MAT as pyrogenicity assay
Among others, the general differentiator between MAT kits is the cell source they make use of. Besides the more commonly used cryopreserved pooled Peripheral Blood Mononuclear Cells (PBMCs), the only other commercialised cell source is the Monocytic Continuous Cell Line – otherwise referred to as ‘Mono-Mac 6’ (MM6) cells. On the feasibility of MM6 cells, the EP (2.6.8) concludes:
EP 2.6.8.
"Monocytic cell lines are appropriate for the detection of bacterial endotoxins but have limited use for the detection of non-endotoxin pyrogens."
The MM6’s unsuitability to be employed too for non-endotoxin detection means similar risk assessments to those required for BET would need to be employed wherever it’s used. In other words, doubling an organisations expenditures on their risk assessments.