Pyrogen Testing

Pyrogen testing has been occurring for the best part of 100 years. In three parts, this page overviews the history of pyrogen testing, what pyrogens are and finally the different assays that are commercially available to test for them – as well as an evaluation of their strengths and weaknesses.

A brief history of pyrogen testing

From as far back as the 19th century, adverse reactions after the administration of parenteral drugs have been logged – at that time described as “injection fever”. It took until 1912 however for Hort and Penfold to 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.

Over time however, the great number of pyrogen tests carried out using rabbits and the increasingly obvious variable sensitivity called for advancements in alternative assays. One such alternative was the bacterial endotoxin test (BET) based on the Limulus Amebocyte Lysate (LAL) from the blood of horseshoe crabs. Becoming commercialised in the 70’s, this test has been widely employed as an assay to detect endotoxin pyrogenic contamination from Gram-negative bacteria –  in particular lipopolysaccharides (LPS) from bacterial cell walls, which are highly resistant against heat.


From the 1980’s onwards, increasing biological complexity in drugs  (e.g. biotechnology and cell therapy products) and their production processes has meant that quality control for pyrogenic contamination has grown equally complex. More complicated production processes bring with them risks of other contaminants – namely Non-Endotoxin Pyrogens (NEPs) – being found in parenteral products. NEPs derive from Gram-positive bacteria or viruses from animal based raw materials (among others) and are not detectable by LAL or other BET based assays.


As a response to the increasing complexity of parenteral drugs; the paucity of pyrogen tests besides the qualitative, human-unspecific RPT; and the innate limitations of BET, in 2016 the European Pharmacopoeia (chapter 5.1.10.) introduced the necessity of a risk assessment for the product, production process and raw materials in respect to pyrogenic contaminations that are undetectable by the bacterial endotoxin test. As of 2010, the Monocyte Activation Test has been pointed to as the compendial method of Pyrogen Detection in the European

Pharmacopeia (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).

Defining pyrogens

What is a pyrogen?

A pyrogen is a substance that induces a rise in temperature in a human or animal. Pyrogens can be stratified broadly into two categories: Exogenous and Endogenous pyrogens. Exogenous pyrogens produce fever after parenteral administration while Endogenous pyrogens like IL-1, IL-6, IL-12 or TNF-α are produced by the body itself as a reaction to contact with exogenous pyrogens. 


Pyrogenic contamination can occur in the production process of pharmaceuticals, biotherapeutics and medical devices – as well as the raw materials used or even during their administration. That being said, the presence of pyrogens could also be a feature intrinsic to the product in question – for example adjuvants in vaccines or synthetic lipopeptides.

Types of exogenous pyrogens

Based on their origin, a number of exogenous pyrogens can be characterised. These are broadly classified into two groups: (1) Endotoxins, and (2) Non-endotoxins.

Endotoxin pyrogens

Comprising components from Gram-negative bacteria, in particular lipopolysaccharides (LPS) from bacterial cell wall, which are highly resistant against heat 

Non-endotoxin pyrogens (NEP)

  • Components of Gram-positive bacteria such as peptidoglycan, lipoteichoic acids and bacterial lipoproteins;

  • Viral pyrogens, in particular virion components from myxoviruses such as influenza;

  • Pyrogens from yeast and fungi like capsular polysaccharide;

  • Pyrogens from non-biological sources such as rubber particles, microscopic plastic particles or metal compounds in elastomers.

Pyrogenic activation of human immune response

Produced in bone marrow, Monocytes are a type of leukocyte or white blood cell involved in innate immunity that fight certain infections and help other white blood cells remove dead or damaged tissues, destroy cancer cells, and regulate immunity against foreign substances. 


Monocytes have cell surface receptors called Pattern Recognition Receptors (PRRs) that recognise antigens. The specific PRRs that recognise pyrogens are called Toll-like Receptors (TLRs). Upon recognition of a pyrogen TLRs activate an immune response through the production of endogenous pyrogens such as cytokines. Cytokines in turn have a direct effect on temperature regulation in the hypothalamus – i.e. cause fever response.

Why should we test for pyrogens?

All parenterally administered drugs or medical devices must be “pyrogen-free”.

This is because exogenous pyrogens can induce fever, inflammatory reactions, multi organ failure and in some cases even death.

Available pyrogen testing methods

Bacterial endotoxin tests (BET) or Limulus amebocyte lysate (LAL) 

Method description 

These are assays that detect endotoxins from Gram-negative bacteria on the basis of clotting reaction of hemolymph in the horseshoe crab’s immune response system.


  • High sensitivity

  • Fast turnaround time

  • Very affordable

  • Simple test protocol to follow


  • Is not a human-specific assay

  • Does not detect non-endotoxin pyrogens

  • Does not independently fulfil mandate of “pyrogen test” for batch release in EP – requires risk assessment → more cost.

  • Cellular or blood products, proteins, lipids, aluminium hydroxide adjuvants (common in vaccines), glucans (false positives) cannot be tested.

  • Very susceptible to interference from conditions

  • Horseshoe crabs die 15% of the time after their blood is drawn for LAL – leading to endangerment of species.


Recombinant Factor C (rFC) endotoxin test


Method description

Based on a rFC, which is a genetically engineered protein that can be activated by endotoxins and in turn produces a fluorescent end product which is quantifiably measurable.



  • Non-animal, In vitro test

  • High sensitivity to Endotoxin pyrogenic contamination

  • Fast turnaround time

  • Very affordable

  • Simple test protocol to follow



  • Is not a human-specific assay

  • Does not detect non-endotoxin pyrogens

  • Does not independently fulfil mandate of “pyrogen test” for batch release in EP – requires risk assessment → more cost.

  • Cellular or blood products, proteins, lipids, aluminium hydroxide adjuvants (common in vaccines) cannot be tested.

  • Very susceptible to interference from conditions (e.g. pH, ionic strength, enzyme activity, endotoxin masking / low endotoxin recovery (LER))

Pyrogen Test (Rabbit Pyrogen Test: RPT)

Method description

Measuring the change in body temperature following injection of test solution on three rabbits.



  • Detection of both endotoxin and non-endotoxin pyrogenic contaminants



  • Significantly lower sensitivity (0.5 EU/mL) than other tests

  • Though responds well to LPS, it is not very responsive to Gram-positive bacteria

  • Does not produce a quantitative results

  • No positive control

  • Can’t be used to test all products (e.g. chemotherapeutics or immunosuppressive agents) 

  • Can’t be used to test human cellular preparations (e.g. blood components and stem cells) 

The Monocyte Activation Test (MAT)

Method description

Monocytes are exposed in tissue culture to the test substance. If the test substance contains pyrogens, these will elicit IL-6 production in the monocytes, which can be detected by an IL-6 ELISA in the culture supernatant.



  • In vitro – no animals are harmed

  • Human specific assay

  • Detects full spectrum of endotoxin and non-endotoxin contaminants

  • Suitable for every kind of parenteral drug or medical device

  • Quantitative read out

  • Most accurate lower limit of detection

  • Highest sensitivity of all pyrogen tests



  • Takes longer than LAL to receive results 

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