In basic terms what is a pyrogen and what is the difference between an endotoxin pyrogen and a non-endotoxin pyrogen? Why is it so important to detect non-endotoxin pyrogens?
Pyrogens are substances that induce a rise in the body temperature of a human being or an animal, to the point of becoming a health concern. The pyrogens that contaminate pharmaceutical products most frequently are lipopolysaccharides (LPS), also known as endotoxins, which are major components of the outer membrane of Gram-negative bacteria. But there are many other pyrogens of microbial origin, including lipoteichoic acid (LTA), a constituent of the cell wall of Grampositive bacteria, particles from viruses and substances from yeasts and fungi. Non-microbial substances can have pyrogenic properties as well, for example rubber particles, microscopic plastic particles or metal compounds in elastomers. Endotoxins therefore constitute a subgroup of pyrogens, albeit the most relevant one for the safety of pharmaceuticals. This is why all other pyrogens are collectively referred to as non-endotoxin pyrogens, a heterogeneous group in terms of chemical structure. However, such non-endotoxin pyrogens can pose risks to patient safety as well. Testing parenteral drugs for the most significant group of pyrogens (i.e., endotoxins) is crucial, testing them for all types of pyrogen is elevating patient safety to a higher level, in particular when you consider that the more complex processes involved in manufacturing today’s biologics and cell therapy products can entail different contamination profiles.
Can you tell us how the regulations regarding the detection and elimination of pyrogens has evolved over the years?
In the late 19th century it was observed that patients can react to injections by developing fever. This led to the development of the rabbit pyrogen test (RPT), which was included in the US Pharmacopeia in 1942 and has since been refined several times. It is still among the most commonly used pyrogen tests. This in vivo test potentially detects all types of pyrogen, including non-endotoxin pyrogens. However, it does have limitations. The RPT is unsuitable for vaccines containing outer membrane vesicles, it generates qualitative rather than quantitative results, and the rabbit models are highly variable. There are also ethical issues: the animals can suffer pain, develop fever, breathing problems, circulatory and organ failure, and even die from fatal shock. This is why alternatives have been developed, one of which is the bacterial endotoxin test (BET) based on the Limulus Amebocyte Lysate (LAL) from the blood of horseshoe crabs. The BET is an in vitro endotoxin assay that has become accepted by the main regulatory drug agencies around the world, including the FDA. Its main drawbacks are that it does not detect non-endotoxin pyrogens and, like the RPT, is based on a non-human reaction.
Knowing the limitations in the RPT and LAL methods, a new in-vitro method was developed as an alternative to the historical methods. The monocyte activation test (MAT) was developed to be an alternative primarily to the RPT and in cases where LAL was not suitable. In 2010 the European Pharmacopeia released a new chapter on the monocyte activation test, revised in 2017 to outline the approved methods and sources of monocytes to be utilized in product specific validation, and to replace the RPT where possible. Additionally, in 2012, the FDA released a Q&A guidance that gives acknowledgement of both MAT and rFC (recombinant factor C) as suitable alternative methods of BET. Looking forward, many hope that a comparable USP chapter will be drafted to compliment the EP chapter that currently exists.
What is a Monocyte Activation Test and why was it such a big step forward in pyrogen detection?
The Monocyte Activation Test (MAT) uses monocytes of three main sources to detect pyrogens. These approved sources include whole human blood (or cryopreserved blood), Peripheral Blood Mononuclear Cells (PBMCs) and Monocytic Continuous Cell lines, such as the MM6 cell line. When the sample to be tested comes into contact with human monocytic cells, the MAT mimics what happens in the human body: in the presence of pyrogens, the monocytes are activated and produce cytokines. These cytokines are then detected using an immunological assay (ELISA) involving specific antibodies and an enzymatic color reaction. The MAT is a big step forward because it overcomes many of the limitations of the RPT and the BET. It is an in vitro test based on a human reaction allowing the detection of both endotoxin and non-endotoxin pyrogens while delivering fully quantitative results. The MAT does not raise the ethical questions that the RPT and, to a lesser degree, the BET – many of the crabs die following the bleeding process – do. The MAT has been a compendial method for pyrogen detection in the European Pharmacopeia since 2010 and also features in the FDA’s “Guidance For Industry – Pyrogen and Endotoxins testing: Questions and Answers” as an alternative to the RPT and should be validated according to USP <1225>. In addition, USP <151> states that “A validated, equivalent in vitro pyrogen or bacterial endotoxin test may be used in place of the in vivo rabbit pyrogen test, where appropriate.”
Please describe how the PyroMAT® system works and how its features and benefits can help pharmaceutical companies meet regulatory guidelines.
The PyroMAT® system uses cryo-preserved Mono-Mac-6 (MM6) human monocytic cells as its source of monocytes. These cells express a variety of toll-like receptors (TLRs) that recognize different microbial ligands. Upon binding, such ligands stimulate their corresponding TLRs, which subsequently initiate signaling cascades that trigger specific immunological responses. MM6 cells respond by producing the cytokine interleukin-6, which is what the PyroMAT® system detects and quantifies. A recent study demonstrated that the various TLRs of the MM6 cells detect a wide range of ligands originating from various Gram-negative and Gram-positive bacteria, fungi, viruses and parasites. The diversity of the TLR family and the specificity of individual TLRs to detect specific ligands support the hypothesis that the human fever reaction can be provoked not only by LPS, but also by many other pyrogenic substances originating from organisms other than Gram-negative bacteria. This, in turn, suggests that the BET is unable to detect many pyrogens that can induce fever in humans, leaving the patient safety risks posed by nonendotoxin pyrogens undetected.
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Looking forward do you see any tightening of regulations regarding pyrogen testing and, if so, how will technology like the PyroMAT® system continue to offer a robust, reliable and standardized method for pyrogen detection.
The main concern is that many products under development now are becoming increasingly more complex and targeted with the dynamic advances in the cell and gene therapy fields. A methodical approach to risk mitigation is necessary, which should include a holistic approach to pyrogen testing. Minimally, a risk assessment should be performed to rule out any inherent pyrogens in the product, and then a decision to be made to continue with a total pyrogen test like MAT, or move to LAL when non-endotoxin pyrogens can be ruled out. Utilizing a product like the PyroMAT® system can be a tool when performing a new product risk assessment, to retroactively evaluate commercialized products, or as a standing release method for validated products. The monocyte activation test and MM6 cell line have been independently validated, and have demonstrated consistency, high sensitivity, and repeatability.