Evidence Against a Bacterial Endotoxin Masking Effect in Biologic Drug Products by Limulus Amebocyte Lysate Detection

Bolden, Jay S.; Claerbout, Mark E.; Miner, Matthew K.; Murphy, Marie; Smith, Kelly R.; Warburton, Rob E.

doi:10.5731/pdajpst.2014.00999

Cited by 13 publications

(13 citation statements)

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“…LPS in native endotoxin is imbedded in cell wall fragments and may be protected from the dispersing effects of chelating buffers and surfactants and is therefore probably not prone to masking effects. Hence, it is argued that the observed masking effects would only represent an artefact emerging from faulty experimental design – as claimed in recent studies performed by Bolden et al 1415…”

mentioning

confidence: 84%

Biological Activity of Masked Endotoxin

Schwarz

Gornicec

Neuper

et al. 2017

Sci Rep

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Low endotoxin recovery (LER) is a recently discovered phenomenon describing the inability of limulus amebocyte lysate (LAL)-based assays to detect lipopolysaccharide (LPS) because of a “masking effect” caused by chelators or detergents commonly used in buffer formulations for medical products and recombinant proteins. This study investigates the masking capacities of different buffer formulations and whether masked endotoxin is biologically active. We show that both naturally occurring endotoxin as well as control standard endotoxin can be affected by LER. Furthermore, whereas masked endotoxin cannot be detected in Factor C based assays, it is still detectable in a cell-based TLR4-NF-κB-luciferase reporter gene assay. Moreover, in primary human monocytes, masked LPS induces the expression of pro-inflammatory cytokines and surface activation markers even at very low concentrations. We therefore conclude that masked LPS is a potent trigger of immune responses, which emphasizes the potential danger of masked LPS, as it may pose a health threat in pharmaceutical products or compromise experimental results.

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mentioning

confidence: 84%

Biological Activity of Masked Endotoxin

Schwarz

Gornicec

Neuper

et al. 2017

Sci Rep

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“…There is a discussion on the difference in LER effects between purified LPS and naturally occurring endotoxin (NOE). NOE is relatively resistant to LER [5,6,9,10]. Structures of NOE are affected by the environment of the growth of the bacteria.…”

Section: (4)mentioning

confidence: 99%

“…The interfering factors test is based on the confirmation of acceptable recovery (50%-200%) in a diluted sample. Samples in LER studies were usually diluted with water for the BET, and the diluted samples were confirmed to overcome interference of the sample by the interfering factors test [5][6][7][8][9][10]. This indicated that the diluted samples were not inhibitory to the activation of LAL, and that endotoxin activity in the diluted samples should be detected by the BET if there was still activity of the spiked endotoxin.…”

Section: Introductionmentioning

confidence: 99%

Mechanism of Low Endotoxin Recovery Caused by a Solution Containing a Chelating Agent and a Detergent

Tsuchiya¹

2019

Immunome Res

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Mechanism of Low Endotoxin Recovery (LER) was investigated by observing the change in particle sizes and activity of endotoxin in LER solutions containing sodium citrate and polysorbate 20. Interestingly, endotoxin aggregates were not dispersed to be smaller particles under LER conditions according to the decease of the activity. This observation was different from previous predictions of the mechanism. Endotoxin aggregates were observed by Dynamic Light Scattering, and activity was measured by the Limulus amebocyte lysate test. Particles with sizes similar to endotoxin aggregates were always observed despite different remaining activity of spiked endotoxin. This observation differed from previously proposed mechanisms for LER that dispersed endotoxin aggregates were smaller in sizes. Based on the findings, a new mechanism of LER is proposed. Purified endotoxin forms non-lamellar cubic structures in water, and the endotoxin aggregates are reinforced by divalent cations. When purified endotoxin is added to LER solutions, divalent cations are removed from outer layer of endotoxin by the chelating agent, and the loss of divalent cations weakens the outside layer of the endotoxin aggregates. The endotoxin aggregation structure is maintained at low temperature, but endotoxin molecules on the surface of the endotoxin aggregates are gradually replaced with detergent molecules at higher temperature. This replacement reduces the surface area of the endotoxin, and cause reduction of activity of the endotoxin aggregates. The apparent sizes of the aggregates are not changed after the replacement.

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“…According to the "3Rs principle" for laboratory animals (Franco & Olsson, 2013), which represents reduction, refinement, and replacement, there is a need to find alternative approaches to test pyrogens. Several international groups have recently developed new in vitro pyrogen testing methods (Daneshian, Guenther, Wendel, Hartung, & Aulock, 2006;Fennrich et al, 2016). However, variation among monocyte donors means that this test requires monocytes from four different individuals, and as acquiring fresh human mononuclear cells can be a problem for many laboratories, this method is not very easy to implement (Burger-Kentischer, Abele, Finkelmeier, Wiesmüller, & Rupp, 2010;Nakagawa, Maeda, & Murai, 2002;Timm, Hansen, Moesby, & Christensen, 2006).…”

Section: Introductionmentioning

confidence: 99%

“…However, variation among monocyte donors means that this test requires monocytes from four different individuals, and as acquiring fresh human mononuclear cells can be a problem for many laboratories, this method is not very easy to implement (Burger-Kentischer, Abele, Finkelmeier, Wiesmüller, & Rupp, 2010;Nakagawa, Maeda, & Murai, 2002;Timm, Hansen, Moesby, & Christensen, 2006). Several international groups have recently developed new in vitro pyrogen testing methods (Daneshian, Guenther, Wendel, Hartung, & Aulock, 2006;Fennrich et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Construction of a sensitive pyrogen‐testing cell model by site‐specific knock‐in of multiple genes

Lei

et al. 2019

Biotech & Bioengineering

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A pyrogen test is crucial for evaluating the safety of drugs and medical equipment, especially those involved in injections. As existing pyrogen tests, including the rabbit pyrogen test, the limulus amoebocyte lysate (LAL) test and the monocyte activation test have limitations, development of new models for pyrogen testing is necessary. Here we develop a sensitive cell model for pyrogen test based on the lipopolysaccharides (LPS) signal pathway. TLR4, MD2, and CD14 play key roles in the LPS-mediated pyrogen reaction. We established a new TLR4/MD2/CD14-specific overexpressing knock-in cell model using the CRISPR/CAS9 technology and homologous recombination to detect LPS. Stimulation of our TLR4/CD14/MD2 knock-in cell line model with LPS leads to the release of the cytokines IL-6 and TNF-alpha, with a detection limit of 0.005 EU/ml, which is greatly lower than the lower limit of 0.015 EU/ml detected by the Tachypleus amebocyte lysate (TAL) assay.

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Evidence Against a Bacterial Endotoxin Masking Effect in Biologic Drug Products by Limulus Amebocyte Lysate Detection

Cited by 13 publications

References 0 publications

Biological Activity of Masked Endotoxin

Biological Activity of Masked Endotoxin

Mechanism of Low Endotoxin Recovery Caused by a Solution Containing a Chelating Agent and a Detergent

Construction of a sensitive pyrogen‐testing cell model by site‐specific knock‐in of multiple genes

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