Pyrogen Testing

Contents



Pyrogen Testing

The detection of pyrogens within parenteral pharmaceutical products and medical devices is a globally mandated safety requirement for manufacturers. Because of considerable shifts in the European Pharmacopoeia (Ph.Eur. 5.1.10) as well as noted limitations of the RPT (rabbit) and LAL (horseshoe crab) methods, MAT testing, in accordance with Ph. Eur. 2.6.30 (July 2016) has emerged as a significant in vitro test to evaluate the existence of pyrogens in such products.

European Pharmacopoeia 8th Edition: Supplement 8.8

  • A revised version of the chapter on pyrogens, which recommends replacing this test by the Monocyte-activation test wherever possible and after product-specific validation to avoid the use of live animals.
  • A revised chapter on the use of the test for bacterial endotoxins, which includes new recommendations on the need to perform risk assessment when using the bacterial endo toxin test as a pyrogenicity test, due to the potential contamination by non-endotoxin pyrogens; and a section on how to set limits for bacterial endotoxins.
  • MAT is endorsed as a suitable element of this risk assessment.



The Monocyte Activation Test

The results from monocyte activation through microbial products. The Monocyte Activation Test (MAT) detects this reaction in vitro, as outlined Briefly, when performing MAT, PBMC (that have been cryopreserved instantly after their isolation form the human body) are exposed in tissue culture to the test substance. If the test substance contains microbial contaminants, these will elicit IL-6 production in the monocytes, which can be detected by an IL-6 ELISA in the culture supernatant.

How RPT works

Schematic representation of the fever reaction in higher vertebrates, including in rabbits and humans.



Principles of MAT

Step 1 - Plating of PBMC in 96 well microtiter plates

Step 2 - Addition of the test substance

Step 3 - Incubation


Representing a sensitive in-vitro bioassay, successful implementation of MAT critically depends on the availability of fully functional monocytes as indicator cells, optimized and pyrogen-free tissue culture conditions, and a sensitive cytokine detection in the culture supernatant. Our experts are available to perform and interpret test results, including product-specific validation and interference matrices, all while documenting and reporting the test procedure to meet regulatory compliances. CTL-MAT is the only company that can help you with any and all these requirements for performing the MAT at your own facility – or we can perform the test for you in our own certified laboratories.

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How RPT works

Schematic representation of the fever reaction in higher vertebrates, including in rabbits and humans. Microbial products (“exogenous pyrogens” enters the body (1) and bind to receptors for “common features of foreign”, so called TLRs on macrophages and monocytes (2). (Monocytes are precursor cells to macrophages, the latter being tissue resident, the former circulating in the blood). The Macrophages and monocytes become activated by the TLR stimulation (3) and respond with cytokine production: among the cytokines these cells produce is IL-6 (4) an "endogenous pyrogen". This IL-6 reaches the brain via the blood stream (5) and is perceived in the thermo regulation center (TRC) (6), being located in the pre-optic area of the hypothalamus. In response, the TRC raises the desired body temperature level, and fever results (7). The RPT measures the natural fever reaction of the body of the experimental animal, in vivo. MAT measures the initial steps, 2-4, in tissue culture, in vitro.

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Principles of MAT

Step 1 - Plating of PBMC in 96 well microtiter plates

A minor sub-population of human PBMC consist of monocytes, elite members of the innate immune system. The function of these cells is to recognize common features of ^ ^ foreign ^ ^ relying on their pattern recognition receptors, also called or toll like receptors (TLRs). In the absence of TLR stimulation, monocytes are in a resting state and do not secret cytokine (illustrated in the figure as the blue cells). Each batch of MAT PBMC offerd by CTL-MAT is thoroughly screened for containing resting monocytes only, thus producing a low cytokine background for the CTL MAT Kit.

Step 2 - Addition of the test substance

If a test substance is contaminated with microbial products, these will bind to TLRs on monocytes, resulting in the activation of these cells. If LPS is present, it will bind to TLR4, LTA ( a constituent of Gram positive bacteria), peptidoglycan (PGN, a constituent of most bacteria), or Zymosan (a constituent of yeast) will bind to TLR2, leading to monocyte activation. As MAT tests sensitively detect microbial contaminants in test substances, we made sure that all constituents of the CTL MAT Kit are void of such, thus producing a low cytokine background for the CTL MAT Kit.

Step 3 - Incubation

Upon TLR engagement in the presence of microbial antigens (contaminations), monocytes become activated (shown in the figure as red cells) and start secreting cytokines including interleukin 6 (IL-6), the primary inducer of the fever reaction in vivo. It takes monocytes approximately 20 h to reach peak level of IL-6 production. To allow for it, the PBMC are cultures with the test substance for 20 h before IL-6 is measured in the superna- tant.For successful MAT assays, therefore, PBMC donors need to be selected who are particularly proficient in IL-6 production. Moreover, the PBMC need to be cryopreserved in a way so they retain their full viability and functionality in spite of the cryopreservation. Each batch of PBMC included as indicator cells in CTL-MAT kits has been carefully selected for unimpaired IL-6 production upon thawing.



MAT vs. RPT

The Monocyte Activation Test (MAT) vs. the Rabbit Pyrogen Test (RPT)

People throughout the world should be able to count on parenteral drugs, cosmetics and biopharmaceuticals to be safe. The same holds true for medical devices. Microbial contamination, however, can happen even under the most stringent manufacturing standards.

Such contaminations can lead to conditions ranging from mild fever to life-threatening septic shock in recipients. This is why regulatory agencies throughout the world mandate "pyrogenicity testing" of every production batch of parenteral drugs and medical devices.

A product's pyrogenicity due to contamination, that is, its property to induce a fever reaction has been tested traditionally by injecting it into rabbits, in the , RPT. While the RPT is ideally suited to detect microbial contaminants of all kinds (constituents of Gram-positive and Gram-negative Bacteria, viruses, fungi and mold), it involves animal testing (see below).

Pyrogens detected by the Rabbit Pyrogen Test (RPT), by the Monocyte Activation Test (MAT)
Pyrogen RPT MAT LAL rFC
Gram-negative bacteria Yes Yes Yes Yes
Gram-0positive bacteria Yes Yes No No
Fungi, Mold Yes Yes No No

elicited in rabbits (and humans) results from monocytes, a subpopulation of white blood cells, are activated in the body through the exposure to microbial products, resulting in these monocytes releasing endogenous cytokines, such as IL-6.IL-6 then stimulates the thermocenter of the brain to raise the body temperature, resulting in fever.

As the mechanism of this fever reaction is in the meantime well understood, it has become possible to model it in cell culture experiments in vitro via the Monocyte Activation Test (MAT) the principle of this test is described As MAT has been proven to be a superior detection system for microbial contaminations compared to the RPT, however without involving animal sacrifice. The European Pharmacopeia has mandated to avoid the RPT whenever possible in favor of in vitro laboratory testing, and in Ph. Eur. 2.6.30 has come up with strict guidelines for the implementation, product-specific validation and reporting for MAT.

MAT has additional advantages over RPT: It's applicable to a greater variety of products than RPT, and furthermore it is more precise, reliable, and cost-effective compared to RPT.

CTL-MAT's Test kit and customer support structure has been established to help realize the goal of maximal drug safety avoiding unnecessary animal cruelty.



MAT vs. LAL

MAT vs. LAL (and recombinant Factor C) Test

White blood cells of the North American horse shoe crab, limulus, contain a protein called Factor C, that agglutinates in the presence of lipopolysaccharides (LPS). Relying on this reaction, the Limulus Amebocyte Lysate test (LAL) has emerged as an in vitro alternative for the RPT to detect contaminations with LPS. The test principle of the LAL test is described Unfortunately, at least one third of the crabs do not survive the bleeding. To avoid unnecessary animal cruelty, the trend is to obtain Factor C not by bleeding of these animals, but by production as a recombinant protein. Irrespective of Factor C's origin, the test principle is the same resulting in a highly sensitive and specific tests for the detection of LPS (and LPS only -other microbial contaminants equally hazardous go undetected (see below). LPS is contained in Gram-negative bacteria, but not in Gram-positive bacteria, in fungi and viruses. Therefore, LAL and Factor C tests fail to detect all these contaminations, except for LPS.

Pyrogens detected by the Rabbit Pyrogen Test (RPT), by the Monocyte Activation Test (MAT)
Pyrogen RPT MAT LAL rFC
Gram-negative bacteria Yes Yes Yes Yes
Gram-0positive bacteria Yes Yes No No
Fungi, Mold Yes Yes No No

LAL Test

The LAL and rFC tests are based on a enzymatic cascade activated by endotoxin in horse shoe crabs.



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LAL Test

The LAL and rFC tests are based on a enzymatic cascade activated by endotoxin in horse shoe crabs. Much like the human blood coagulation- or complement system can become activated by invading bacteria, also horse shoe crabs possess innate immune reactivity to Gram-negative bacteria: endotoxin activates the conversion of Factor C into its activated variant, Factor C*. The LAL detects the resulting clotting reaction in limulus blood (hemolymph) either via a gel clotting- , or a turbimetric-, or a chromogenic read-out. In the rFc test, recombinant Factor C is used whereby its activation by endotoxin generates a fluorescent end product that can be detected.

MAT detects all the above microbial contaminants. Monocytes rely on a family of pattern recognition receptors, called toll-like receptors to identify such targets and to eliminate them by phagocytosis. LPS engages TLR-4 on monocytes, triggering them to produce IL-6 (thus, like LAL, MAT also detects LPS). Lipoteichoic acid (LTA), a constituent of Gram-positive bacteria, triggers TLR-2 on monocytes, as does peptidoglycan (PGN), a constituent of most bacteria, or Zymosan, a constituent of yeast). By expressing 9 different types of TLRs as known today, monocytes are well equipped to recognize most microbial agents. For a summary of TLRs and their respective microbial ligands

TLRs and their ligands

Cells of the innate immune system, such as macrophages and monocytes possess receptors for "common features of foreign".



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TLRs and their ligands

Cells of the innate immune system, such as macrophages and monocytes possess receptors for “common features of foreign”. These receptors belong to the toll-like receptor (TLR) family. To date, ten TLR receptor family members are known, each member binding a different molecule-type that is characteristic for microbial antigens. The respective ligands for the individual TLR members are specified above each TLR in the figure. If a TLR binds its ligand, that results in activation of the macrophage/monocyte, inducing the cell to secrete cytokines such as the ‘endogenous pyrogen”, IL-6. Through this mechanism, by activating “endogenous pyrogen”/IL6 production microbial products induce the fever reaction, that is, become “exogenous pyrogens”.

Unlike the LAL test (and recombinant Factor C assay), MAT is uniquely suited to detect microbial contaminations of all kinds beyond LPS. Representative data showing detection of key microbial contaminants beyond LPS by MAT are shown .

Pyrogen Detection

CTL-MAT kits detect pyrogens of various types, beyond LPS.



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Pyrogen Detection

CTL-MAT kits detect pyrogens of various types, beyond LPS. The microbial substances specified in each panel (with the TLRs they bind specified in parenthesis) were tested in CTL MAT assays at the concentrations specified at the respective X axes. The concentration of IL-6 detected in the culture supernatant after 20 h incubation of the PBMC with these substances is shown on the Y axis.

Like the MAT, the LAL and the rFC are compendial methods, i.e., they need to undergo product-specific validation. As the MAT is accepted as an animal- free quantifiable method for pyrogen testing, the Eu. Ph. requires MAT, instead of the RPT to be used for the validation of LAL and rFC.