Pyrogen testing

Pyrogen testing

Parenteral administered pharmaceutical products must be free of pyrogenic (fever-inducing) contamination as these substances may induce life-threatening systemic inflammation in the recipient. Pyrogens are classified in two groups; 1) endotoxins, originating from gram-negative bacteria and 2) Non-Endotoxin Pyrogens (NEPs), which can originate from gram-positive bacteria, viruses or fungi. Two widely used pyrogen test methods are the rabbit pyrogen test (RPT) and the bacterial endotoxin test (BET) based on Limulus Amebocyte Lysate (LAL).

Both tests have their limitations. The RPT can miss human-specific pyrogens, does not allow for quantification and requires the use of animals. The BET requires the use of blood from the horseshoe crab and does not detect NEPs.

Overview of pyrogen tests and their characteristics

Pyrogen tests		BET	RPT	MAT
Non-animal, human-based test		-	-	+
Detection of endotoxin		+	+	+
Detection of NEPs	Human-specific	-	-	+
	Yeasts & molds	-	+	+
	Virus	-	+/-	+

In recent years an alternative in vitro pyrogen test, the Monocyte Activation Test (MAT), has been developed to detect and quantify endotoxin and NEP contaminations. The assay is based on the human immune response by measuring cytokine production of human peripheral blood mononuclear cells (PBMC). Monocytes are the main cells within the PBMC population that produce cytokines upon the presence of pyrogens, hence the name Monocyte Activation Test.

The principle of the test is that PBMCs are incubated with the test sample, controls, and depending on the method, an endotoxin standard curve. Following incubation at 37°C, the cell culture supernatant is collected and analyzed for the presence of cytokines, e.g. interleukin-6 (IL-6), by enzyme-linked immunosorbent assay (ELISA). Since 2010, the European Pharmacopoeia (EP) considers the MAT as a replacement for the rabbit pyrogen test (EP chapter 2.6.30).

The benefits of MAT as a pyrogen test:

• Mimicks the humane immune response
• Detects all pyrogens relevant to humans
• Does not require the use of animals
• Can analyse pyrogenic activity in more complex pharmaceuticals, like:
  • Blood-derived products
  • Cell-derived products
  • Biologics and vaccines

MAT relevant for

• Pharmaceutical companies
• Biotech companies
• Medical device companies
• Pharmacies performing in-house pyrogen testing
• Test service and diagnostic laboratories
• Clinical Research Organizations (CRO)