Estelle Zelter- Global Product Manager, Pyrogens, Alternative and Rapid Sterility testing, Millipore SAS, Molsheim, France, An affiliate of Merck KGaA, Darmstadt, Germany; Manjula Aysola- Senior Regulatory Consultant, Contract Testing Services, EMD Millipore Corp., Burlington, USA, An affiliate of Merck KGaA, Darmstadt, Germany; Anne Baumstummler, Ph.D.- Director, Strategic Marketing, BioMonitoring Pharma segment, Millipore SAS, Molsheim, France, An affiliate of Merck KGaA, Darmstadt, Germany
Driven by the 3Rs principle to replace, reduce and refine the use of animals in the testing of pharmaceuticals, the European Pharmacopoeia Commission (EPC) announced in June 2021 that it would put an end to the rabbit pyrogen test (RPT) within five years. This suggests that the EPC views in-vitro techniques to be reliable substitutes to test parenteral preparations for the presence of endotoxins and non-endotoxin pyrogens. An expert roundtable discusses the move and its potential implications for pyrogen testing in the USA.
Did the EPC’s announcement on the RPT come as a surprise to you?
Estelle Zelter: Not really. Obviously, to replace an animal test like the RPT, you have to have a non-animal alternative that can get you equivalent or better test results. This is what the monocyte activation test, or MAT, can do. It is an in-vitro pyrogen test that has been compendial in Europe since 2010, when chapter 2.6.30 was added to the EP. Its introduction could be seen as an early indication that further steps would be taken to reduce animal use in pyrogen testing.
Is the United States Pharmacopeia likely to follow suit?
Manjula Aysola: As things stand there are no USP chapters describing any in-vitro tests that cover the entire range of pyrogens. In principle, however, the leading pharmacopoeias are committed to harmonizing their standards, so we will probably see changes to that effect sooner or later. USP contains the chapter 85 on the bacterial endotoxins test, which is based on LAL, the amoebocyte lysate from the endangered horseshoe crab, and involves drawing blood from the living animal. The recombinant Factor C in-vitro test, considered a compendial test in the European Pharmacopeia, can be regarded as a non-animal alternative to the LAL-based test, and it ticks all the boxes for animal welfare. But, like the LAL test, it detects only endotoxins, the lipopolysaccharides of Gram-negative bacteria. It is not designed to test for non-endotoxin pyrogens such as lipoteichoic acid and peptidoglycans from Gram-positive bacteria, certain components of viruses and fungi, and non-biological substances that can cause inflammation in patients.
Isn’t it sufficient to test injectables for bacterial endotoxins?
Anne Baumstummler: In both Europe and the US, manufacturers are expected to perform a risk assessment of each process. In the course of this assessment they must consider all factors that could lead to the uptake of pyrogens that the endotoxin test can’t detect. If the presence of non-endotoxin pyrogens cannot be ruled out, testing only for endotoxins won’t be appropriate, and pyrogen testing has to be performed. At the joint EDQM-EPAA event ‘The future of pyrogenicity testing: phasing out the rabbit pyrogen test’ in February representatives of various pharmacopeias, including the USP, agreed in principle that animal use for pyrogen testing should end. With the RPT on the way out in Europe, and the possibility, or even probability, that the US will embark on a similar course, now is the right time to look at the options going forward. As it is based on blood cells of humans, the MAT is a better predictor than rabbits of a drug’s pyrogenic potential in humans.
Manjula Aysola: I would like to stress that the transition is no vague ambition. Last September the EPC published an extensive strategy paper on the upcoming changes to pyrogen testing, including timelines, with July 2026 envisaged for when the general chapter 2.6.8 on the RPT will be deleted. And only a few weeks ago, a draft for the new general chapter 5.1.13 on pyrogenicity as well as no fewer than 58 revised texts concerning the move away from the RPT were published, and have been up for commenting. So, make no mistake, it’s happening.
What’s involved in implementing the MAT test?
Anne Baumstummler: The MAT is compendial in Europe, so manufacturers there do not need to perform full method validation, only product-specific validation, in other words qualification. In the US, where it is not yet accepted as a compendial method, a full method validation according to USP chapter 1225 guidelines will be required for later stage product development or for commercial products. The regulators are open to the use of the MAT and they will accept it for release testing once validated.
Estelle Zelter: We launched our own MAT, the PyroMAT® system based on the MonoMac 6 human cell line, in 2018 after a complete validation according to EP 2.6.30, using the USP chapter 1225 approach. It is being routinely used in industry, and we have a wealth of information, data and experience for customers to call upon. In addition, we offer expert services, including feasibility studies, training and validation support, to reduce the work burden involved and pave the way for a smooth path to implementation.
Learn more about the monocyte activation test (MAT) for pyrogen detection in injectables
SigmaAldrich.com/PyroMAT
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