Determination of Interference DuringIn VitroPyrogen Detection: Development and Characterization of a Cell-Based Assay

Abstract

Contamination of pharmaceutical products and medical devices with pyrogens such as endotoxins is the most common cause of systemic inflammation and, in worst cases, of septic shock. Thus, quantification of pyrogens is crucial. The limulus amebocyte lysate (LAL)-based assays are the reference tests for in vitro endotoxin detection, in association with the in vivo rabbit pyrogen test (RPT), according to European Pharmacopoeia (EP 2.6.14), and U.S. Pharmacopoeia (USP <85>). However, several substances interfere with LAL assay, while RPT is not accurate, not quantitative, and raises ethical limits. Biological assays, as monocyte activation tests, have been developed and included in European Pharmacopoeia (EP 7.0; 04/2010:20630) guidelines as an alternative to RPT and proved relevant to the febrile reaction in vivo. Because this reaction is carried out by endogenous mediators under the transcriptional control of nuclear factor-kappaB (NF-kappaB), we sought to determine whether a NF-kappaB reporter-gene assay, based on MonoMac-6 (MM6) cells, could reconcile the basic mechanism of innate immune response with the relevance of monocytoid cell lines to the organism reaction to endotoxins. This article describes both optimization and characterization of the reporter cells-based assay, which overall proved the linearity, accuracy, and precision of the test, and demonstrated the sensitivity of the assay to 0.24 EU/mL endotoxin, close to the pyrogenic threshold in humans. Moreover, the assay was experimentally compared to the LAL test in the evaluation of selected interfering samples. The good performance of the MM6 reporter test demonstrates the suitability of this assay to evaluate interfering or false-positive samples.

Biospecimen Science of Blood for Peripheral Blood Mononuclear Cell (PBMC) Functional Applications

“…Most of these assays are performed in non-regulated preclinical research laboratories. However, at least one regulated functional application has recently emerged: the monocyte activation test (MAT) for detection and quantification of pyrogenic contamination in pharmaceuticals [9]. Similarly, PBMCs are used to detect unwanted intrinsic proinflammatory activities of different biological therapeutics through cytokine (IL-6, IL-1β, TNF-α) release [10].…”

Endotoxin contamination of engineered nanomaterials: Overcoming the hurdles associated with endotoxin testing

“…Moreover, the MAT does not quantify endotoxin levels directly but only the induction of proinflammatory cytokine expression as a marker for fever (Dobrovolskaia et al, 2014; Yang Li, Fujita, & Boraschi, 2017). The RPT, by contrast, overcomes many of the interference issues associated with in vitro assays; however, it is also not endotoxin specific, and nanomaterials that are inherently immune stimulatory, immunosuppressive, or carry cytotoxic drugs may interfere in this case also (Jin et al, 2018; Palma et al, 2017).…”

An expert review on current approaches for endotoxin detection in various biological products

“…So, the permanent gap between the observed, and expected fever response in rabbits comparing to humans is fundamental, (4) The rabbit pyrogen test cannot detect endotoxin in LAL reagent water (water for reconstitution) which may contain a very low EL. Radiopharmaceutical products cannot be tested by the rabbit pyrogen tests [44,45].…”