25 Years Alternative Pyrogen Testing: Where do we stand? A Retrospect and Outlook

The ECA and its members have been involved in endotoxin and pyrogen testing for many years, organizing conferences and seminars and expert meetings on these topics. At a well-received conference in Bacharach on the subject of alternative pyrogen testing conducted in 2006 already, the possibilities of the monocyte activation test (MAT) were controversially discussed. At that time, the publication of Hartung and Wendel on whole blood pyrogen testing had already been 10 years old. At the follow-up conference in Berlin in 2009, the various developers of the three MAT methods A (quantitative test), B (semi-quantitative test) and C ("reference lot comparison test") were still engaged in discussions as to which of the tests would be the "best". Its establishment in daily practice was still a long way off, and most authorities and pharmacopoeias had only taken up this topic to a limited extent. At the EU level, activities to establish MAT in the pharmacopoeia were started in 2005 - however, not as a "must" but only as a possible alternative to the classical pyrogen test and without a separate chapter. At the end of 2009, the EMA Guideline "Replacement of rabbit pyrogen testing by an alternative test for plasma derived medicinal products" came into force and in 2010 the MAT was introduced into the European Pharmacopoeia (Ph. Eur. 2.6.30) as an alternative to the rabbit test. Then, in 2014, there was a revision of Ph. Eur. chapter 5.1.10. on testing for bacterial endotoxins, and in 2017 there was a revision of MAT chapter 2.6.30.

It was not until there was a significant increase in pressure within the EU to reduce animal testing by as much as possible and the subsequent reduction in approvals for the rabbit pyrogen test that there was a real push, both in terms of MAT test providers and the number of pharmaceutical manufacturers using it.

Where are we?

A recent article by Thomas Hartung of the Johns Hopkins Bloomberg School of Public Health on the MAT's 25th anniversary now provides the opportunity to pick up this topic again. He writes in his abstract:

"The whole blood pyrogen test invented 25 years ago, and its variant based on cryo-preserved blood one year later, brought momentum into the field of pyrogen testing, which, despite the broad application of the Limulus amebocyte lysate (LAL) assay, aka bacterial endotoxin test (BET), consumed several hundred thousand rabbits per year world-wide. The resulting international validation and lengthy acceptance and implementation process of what are called now monocyte activation tests (MATs) finally is impacting on animal numbers – at least in Europe – reducing them by more than 70% and counting. The author sees no reason for continuing any regulatory rabbit testing for pyrogens except the lack of acceptance of MATs in some regions of the world. The availability of MATs has opened also the discussion about the shortcomings of LAL/BET, namely its restriction to Gram-negative pyrogens, non-reflection of the potency of these in humans, interference and masking by many products, and animal welfare concerns for horseshoe crabs. The obvious advantages of MATs in all these respects should lead to a shift from LAL/BET to MATs. We are starting to see this for vac­cines and medical devices, but other areas like safety testing of blood transfusions, cell therapies and nanomaterials, and the assessment of air-borne pyrogens still need to grasp the opportunity provided by MATs. While the different MATs can jointly serve these needs, the whole blood MAT has some advantages as discussed here."

Wher are we going?

And at this point it is the question, why it took 25 years for many countries in Europe and some others beyond to accept MAT as a replacement of or at least as an alternative method for the rabbit pyrogen test. What are the reasons that lead to this long process?

Now, one of the learnings from this can possibly be that similar issues can be avoided by discussing strategies to support new developments like alternative (rapid) methods, establishing real-time or online monitoring systems.  Because new methods often open up new possibilities and can be more sensitive, faster and more precise. But the results therefore do not always correspond with the results (the results are often given in other units) of the established methods and the limits and specifications in the guidelines and pharmacopoeias based on them. And revisions of the same - to bring them up to the current state of science and technology - are usually lengthy. Therefore, the use of the methods is often limited to an additive use beside the existing tests. Or the implementation as an alternative is disproportionately costly, because each user has to prove the correlation or comparability between the results of the "old" and the new, alternative method himself.

Digitalization, the experience of accelerated procedures for revisions and improving communication between science, industry and authorities might help to establish new, alternative methods much faster.The ECA with its Pharmaceutical Microbiology Working Group is trying to contribute to this.