The future of pyrogenicity testing: Phasing out the rabbit pyrogen test. A meeting report

Cirefice, Gwenaël; Schütte, Katrin; Spreitzer, Ingo; Charton, Emmanuelle; Shaid, Shahjahan; Viviani, Laura; Rubbrecht, Michelle; Manou, Irene

doi:10.1016/j.biologicals.2023.101702

Cited by 6 publications

(4 citation statements)

References 9 publications

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“…RPT cannot truly reflect the human response to pyrogens and requires considerable manpower and materials, leading to a high cost, so it is gradually becoming obsolete. 37 – 39 BET is simple and economical. However, BET can only detect bacterial endotoxins from gram-negative bacteria, 40 which is often interfered with when used to test solutions with a high protein content.…”

Section: Discussionmentioning

confidence: 99%

A novel alternative for pyrogen detection based on a transgenic cell line

He,

Yu,

et al. 2024

Sig Transduct Target Ther

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Pyrogen, often as a contaminant, is a key indicator affecting the safety of almost all parenteral drugs (including biologicals, chemicals, traditional Chinese medicines and medical devices). It has become a goal to completely replace the in vivo rabbit pyrogen test by using the in vitro pyrogen test based on the promoted ‘reduction, replacement and refinement’ principle, which has been highly considered by regulatory agencies from different countries. We used NF-κB, a central signalling molecule mediating inflammatory responses, as a pyrogenic marker and the monocyte line THP-1 transfected with a luciferase reporter gene regulated by NF-κB as an in vitro model to detect pyrogens by measuring the intensity of a fluorescence signal. Here, we show that this test can quantitatively and sensitively detect endotoxin (lipopolysaccharide from different strains) and nonendotoxin (lipoteichoic acid, zymosan, peptidoglycan, lectin and glucan), has good stability in terms of NF-κB activity and cell phenotypes at 39 cell passages and can be applied to detect pyrogens in biologicals (group A & C meningococcal polysaccharide vaccine; basiliximab; rabies vaccine (Vero cells) for human use, freeze-dried; Japanese encephalitis vaccine (Vero cells), inactivated; insulin aspart injection; human albumin; recombinant human erythropoietin injection (CHO Cell)). The within-laboratory reproducibility of the test in three independent laboratories was 85%, 80% and 80% and the interlaboratory reproducibility among laboratories was 83.3%, 95.6% and 86.7%. The sensitivity (true positive rate) and specificity (true negative rate) of the test were 89.9% and 90.9%, respectively. In summary, the test provides a novel alternative for pyrogen detection.

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Section: Discussionmentioning

confidence: 99%

A novel alternative for pyrogen detection based on a transgenic cell line

He,

Yu,

et al. 2024

Sig Transduct Target Ther

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“…For ethical reasons, the in vivo rabbit pyrogenic test (RPT) will disappear in favor of in vitro tests [47]. However, all techniques developed to assay endotoxins and replace this test have their benefits and disadvantages.…”

Section: Discussionmentioning

confidence: 99%

“…This method is very sensitive, and the rabbit model has been shown to be the closest species to humans in endotoxin response. However, non-animal tests will soon replace this test [47].…”

Section: Introductionmentioning

confidence: 99%

Diversity, Complexity, and Specificity of Bacterial Lipopolysaccharide (LPS) Structures Impacting Their Detection and Quantification

Dardelle,

Phelip,

Darabi

et al. 2024

IJMS

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Endotoxins are toxic lipopolysaccharides (LPSs), extending from the outer membrane of Gram-negative bacteria and notorious for their toxicity and deleterious effects. The comparison of different LPSs, isolated from various Gram-negative bacteria, shows a global similar architecture corresponding to a glycolipid lipid A moiety, a core oligosaccharide, and outermost long O-chain polysaccharides with molecular weights from 2 to 20 kDa. LPSs display high diversity and specificity among genera and species, and each bacterium contains a unique set of LPS structures, constituting its protective external barrier. Some LPSs are not toxic due to their particular structures. Different, well-characterized, and highly purified LPSs were used in this work to determine endotoxin detection rules and identify their impact on the host. Endotoxin detection is a major task to ensure the safety of human health, especially in the pharma and food sectors. Here, we describe the impact of different LPS structures obtained under different bacterial growth conditions on selective LPS detection methods such as LAL, HEK-blue TLR-4, LC-MS2, and MALDI-MS. In these various assays, LPSs were shown to respond differently, mainly attributable to their lipid A structures, their fatty acid numbers and chain lengths, the presence of phosphate groups, and their possible substitutions.

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“…Current efforts by the European Pharmacopeia (EP), the European Medical Agency, the World health organization (WHO), and several National Release authorities like German Paul Ehrlich-Institute (PEI, Germany), NIBSC (UK), NOMA (Norway), ISS (Italy) and others, favor the future use of the MAT for drug release and to ban the use of animals. The EP decided to erase the RPT by 2026 [9], replacing it with the MAT. Current sources of macrophages however, challenge the standardization and broad industrial applicability of this in vitro test.…”

Section: Introductionmentioning

confidence: 99%

Sensor macrophages derived from human induced pluripotent stem cells to assess pyrogenic contaminations in parenteral drugs

Abdin,

Mansel,

Hashtchin

et al. 2024

Biofabrication

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Ensuring the safety of parenteral drugs before injection into patients is of utmost importance. New regulations around the globe and the need to refrain from using animals however, have highlighted the need for new cell sources to be used in next-generation bioassays to detect the entire spectrum of possible contaminating pyrogens. Given the current drawbacks of the Monocyte-Activation-Test (MAT) with respect to the use of primary peripheral blood mono-nuclear cells (PBMCs) or the use of monocytic cell lines, we here demonstrate the manufacturing of sensor monocyte/macrophages from human induced pluripotent stem cells (iMonoMac), which are fully defined and superior to current cell products. Using a modern and scalable manufactering platform, iMonoMac showed typical macrophage-like morphology and stained positive for several toll like receptor (TLRs) such as TLR-2, TLR-5, TLR-4. Furthermore, iMonoMacs derived from the same donor were sensitive to endotoxins, non-endotoxins, and process related pyrogens at a high dynamic range and across different cellular densities. Of note, iMonoMac showed increased sensitivity and reactivity to a broad range of pyrogens, demonstrated by the detection of IL-6 at low concentrations of WHO LPS and MALP-2 which could not be reached using the current MAT cell sources. To further advance the system, iMonoMac or genetically engineered iMonoMac with NF-κB-luciferase reporter cassette could reveal a specific activation response while correlating to the classical detection method employing ELISA to measure cytokine secretion. Thus, we present a valuable cellular tool to assess parenteral drugs safety, facilitating the future acceptance and design of regulatory-approved bioassays. 

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The future of pyrogenicity testing: Phasing out the rabbit pyrogen test. A meeting report

Cited by 6 publications

References 9 publications

A novel alternative for pyrogen detection based on a transgenic cell line

A novel alternative for pyrogen detection based on a transgenic cell line

Diversity, Complexity, and Specificity of Bacterial Lipopolysaccharide (LPS) Structures Impacting Their Detection and Quantification

Sensor macrophages derived from human induced pluripotent stem cells to assess pyrogenic contaminations in parenteral drugs

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