Pyrogen Testing - General Context
Pyrogens are made up of a chemically heterogeneous group of compounds that originate from bacteria, viruses, yeast and molds or the host itself. Pyrogens produce a febrile fever response in humans and animals. Pyrogens can be stratified into two categories: exogenous and endogenous 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 result in cytokine release. Endogenous pyrogens are produced by the body when in contact with exogenous pyrogens and produce a febrile fever response.
Endotoxin pyrogenic contaminants derive from Gram-negative bacteria (lipopolysaccharides (LPS)) – that are found on bacterial cell walls, and are very resistant to heat. Non-endotoxin pyrogenic contaminants derive from Gram-positive bacteria (peptidoglycan, lipoteichoic acids and bacterial lipoproteins), viruses (virion components deriving from myxoviruses like influenza), yeast & molds (capsular polysaccharides) as well as from non-biological sources (rubbers, plastics or metals).
Endotoxin pyrogenic contaminants are measured via the well-known bacterial endotoxin test (BET). BET is a significant part of the workload in most microbiologic laboratories involved with the quality control of parenterals and medical devices. BET testing is based upon the Limulus Amebocyte Lysate (LAL), animal derived. The LAL assay is used to detect endotoxins associated with Gram-negative bacteria. Sustainable BET is now made possible using modern biotechnology approaches that developed recombinant cascade reagents using the same LAL cascade as traditional LAL reagent, while eliminating the potential for (1à3)-β-D-Glucans cross reactivity. Recombinant cascade reagents can be used for a wide variety of endotoxin tests, ranging from standard water testing to intrathecal products and products requiring high dilutions to overcome interference.
Both the LAL and recombinant cascade tests are based on enzymatic reactions which render them unsuitable for biologics. Indeed, biologics suffer from the potential presence of non-endotoxin pyrogens which exclude the sole use of LAL or recombinant reagents for testing such products. The ever-increasing number of biologics under development and on the market, made regulatory authorities adapt a novel testing approach. The only suitable assay for biologics is MAT, Monocytes Activation Test. Unlike the LAL and recombinant cascade reagents, the MAT pyrogen test is the assay least susceptible to interfering factors, including any LER effects. From a regulatory point, chapter EP 2.6.8 (July 2016) recommends to replace the Rabbit Pyrogen Test by MAT wherever possible and after product-specific validation. Chapter EP 5.1.10 (January 2017) stipulates that MAT is a suitable method to use to rule out the presence of non-endotoxin pyrogens in substances or products. The MAT as such is describes in Chapter EP2.6.30.
CellMadeTM Laboratories established a pyrogen detection platform based upon LAL chromogenic method and a recombinant cascade approach. CellMadeTM Laboratories also established MAT. Our excellent know-how and proven track-record in cell biology make us the ideal partner to accompany you in your pyrogen monitoring plan.