What is a pyrogen?
Pyrogens are made up of a chemically heterogeneous group of compounds (which originate from bacteria, viruses, fungi or the host itself), that induce a rise in temperature (febrile response) in a human or animal. Pyrogens can be stratified broadly into two categories: exogenous and endogenous pyrogens.
Ex.
Exogenous
pyrogens
Exogenous pyrogens are external to the body and are recognized by the toll-like receptors (TLRs) of monocytes, leading to the activation of the signaling pathways and a cytokine release.
En.
Endogenous pyrogens
Endogenous pyrogens are produced by the body when in contact with exogenous pyrogens (e.g. IL-6) and produce a febrile fever response.
Diversity of exogenous pyrogens
Endotoxin pyrogenic contaminants
Derive from Gram-negative bacteria – e.g. lipopolysaccharides (LPS) – that are found on bacterial cell walls, and are characteristically very resistant to heat.
Non-endotoxin pyrogenic contaminants
Derive from:
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Gram-positive bacteria (e.g. peptidoglycan, lipoteichoic acids and bacterial lipoproteins)
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Viruses (e.g. virion components deriving from myxoviruses like influenza)
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Yeast & funghi (e.g capsular polysaccharide)
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Non-biological sources (e.g. rubber, plastic or metal)
The history of pyrogen testing
1910's: Rise of the Rabbit Pyrogen Test
In 1912, Hort and Penfold formally identify the agents at the root of the fever as “pyrogens”. Hort and Penfold also designed the first – and still most commonly used – pyrogenicity assay which saw the substance material injected into rabbits.
1970s: Breakthrough with BET pyrogen test
Shortcomings of RPT in relation to human specificity, sensitivity and non-quantifiable results led to innovation and gradual adoption of bacterial endotoxin test (BET) based on on the Limulus Amebocyte Lysate (LAL) in the 1970s.
1980's: Rising NEP significance in pyrogen testing
Increasing biological complexity in drugs (e.g. biotechnology and cell therapy products) and their production processes meant QC for pyrogenic contamination has grown equally complex. Risks of other pyrogenic contaminants – namely non-endotoxin pyrogens (NEPs) become significant. Not detectable by BET.
2010's: E.P. introduces MAT as recommended pyrogen test
MAT was pointed to as the compendial method of pyrogen detection in the European Pharmacopoeia (chapter 2.6.30.) and since the 2016 revision, recommendations have been given to replace tests on rabbits with the MAT, wherever possible and after product specific validation (EP 2.6.8., Rev. July 2016).
"What do international pharmaceutical regulatory bodies have to say about pyrogen testing?"
Testing the degree of pyrogenic contamination of sterile parenteral drugs and medical devices is a ubiquitously mandated quality control measure by standards defined by international regulatory bodies including the European Pharmacopeia (EP), European GMP – Annex 1, USP 151, FDA Guidance for industry – Sterile Drug Product produced by aseptic Processing – Current Good Manufacturing Practice.
Can you tell me —
Why is pyrogen testing mandated?
At sufficient levels of concentration and biological activity, exogenous pyrogens that enter a human’s bloodstream can cause fever, diarrhea, vomiting, and fatal endotoxic shock.
How does pyrogenic contamination of pharmaceutics occur?
Occurs in materials, production processes or administration of parenterals. Pyrogenicity could also be intrinsic to the product e.g. adjuvants in vaccines or synthetic lipopeptides.
Why not solely test for the more common "endotoxins"?
NEP are increasingly becoming important to test for – specifically, as research findings continue to point to them as causes of human adverse reactions including pains, redness, shivering and fever.
Is there really any evidence for NEP contamination beyond theory?
Yes, a major pharmaceutical recently reported a case study that found adverse effects among patients owing to NEP contamination. Once it implemented MAT, reported number of adverse effects of the drug significantly fell.
Pyrogen testing
methods
The following outlines the methods currently available for pyrogen testing. While the LAL and rFC detect solely endotoxin pyrogens, the RPT and MAT detect both endotoxin and non-endotoxin pyrogenic contaminations.
PYROGEN TEST 1
Limulus Amebocyte Lysate (LAL) Test
Based on a clotting reaction of the hemolymph derived from the horseshoe crab. Three variations methodology: gel-clot, turbidimetric, and chromogenic.
PYROGEN TEST 2
Recombinant Factor C (rFC) Test
A non-animal mimic of the LAL, rFC is a genetically engineered protein, which is activated by endotoxins to produce a fluorescent end product which is quantifiable.
PYROGEN TEST 3
Rabbit Pyrogen Test (RPT)
The Rabbit Pyrogen Test method involves measuring the possible rise in temperature of 3 rabbits following the intravenous injection of a test solution in each rabbit.
PYROGEN TEST 4
Monocyte Activation Test
Sample incubated in human monocytes which produce cytokines/interleukins (IL) if pyrogens are present, that are detected in an immunological assay (ELISA).
Pyrogen test criteria
Limulus Amebocyte Lysate (LAL) Test
Recombinant Factor C (rFC)
Test
Rabbit
Pyrogen Test (RPT)
Monocyte Activation Test (MAT)
Human specific
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✘
✘
✔
Detects endotoxins
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✔
✔
✔
Detects non-endotoxin pyrogens
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✘
✔
✔
Lower limit of detection (LoD) (EU/ml)
0.005
0.005
0.5
0.1 – 0.004
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✘
✔
✔
Low susceptibility to interfering factors
Delivers quantifiable results
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✔
✘
✔
Proficient in testing biologics
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✘
✔
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Avoids animal suffering
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✔
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In-vitro assay
✔
✔
✘
✔
How does the EP regard MAT as a pyrogen test?
EP 2.6.8 Pyrogens: Recommends to replace the Rabbit Pyrogen Test by MAT (2.6.30) wherever possible after product-specific validation (EP 2.6.8, July 2016).
EP 5.1.10 Guidelines for using the test for bacterial endotoxins specifies: "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" (EP 5.1.10, January 2017).
"We've started using MAT as the pyrogen test for all our parenterals. Since doing so, we have a better tool to overcome interfering factors, test products with LER effects, detect synergistic effects of NEPs and safely release products with the very lowest contaminate limit concentrations."
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How can I —
Pyrogen test biologic products
The LAL and rFC are based on enzymatic reactions which render them unsuitable for biologics, while RPT is being phased out. The only suitable assay is the biological, MAT.
Pyrogen test products with low contaminate limit concentrations
Releasing products with low CLCs requires an assay with a very low lower limit of detection (LoD). The assay with the very lowest LoD is MAT BioTech's CTL-MAT kit.
Pyrogen test products with low endotoxin recovery effects
Unlike the LAL and rFC, the MAT pyrogen test is the assay least susceptible to interfering factors, including any LER effects.
Pyrogen test to combat synergistic effects
The biological rather than enzymatic reaction that MAT is based on means that it can reliably detect possible synergies between endotoxins and NEPs in your parenterals.
