Effect of different sample treatment methods on Low Endotoxin Recovery Phenomenon

Bu, Ruixuan; Deng, Xinren; Cao, Yuan; Jin, Jiaqi; Mai, Binliang; Meng, Ke; Liu, Xiaohui; Chi, Jen-Chih; Zhang, Yujie; Qiu, Frank

doi:10.1016/j.mimet.2021.106241

Cited by 6 publications

(5 citation statements)

References 16 publications

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“…However, the clotting enzyme can also be activated by other substances, such as (1→3)-β-d-glucan, leading to false positive results. Additionally, its sensitivity can be reduced by the type and source of LPS being detected, by the sample processing, and by the presence of chelating agents, antibodies, LBP, cationic proteins, surfactants, as well as blood proteins [9,90,144,[149][150][151][152][153][154]. Thus, new assays were developed to overcome those limitations, such as the EndoLISA.…”

Section: Limitations Of Detection Assaysmentioning

confidence: 99%

“…Despite many advantages, its broad sensitivity (0.05-500 EU/mL), its superior robustness in terms of endotoxin spike recovery under different stress-inducing conditions, its insensitivity to glucan contaminations, and the use of animal-friendly recombinant proteins, EndoLISA has some major drawbacks. It can only detect LPS in liquid samples [157] and it is still affected by the presence of chelating agents similar to a conventional LAL assay [90,153,154]. This development was followed by other ELISA-based assays that require LPS to be adsorbed to plastic wells or to be detected by different capture proteins, such as monoclonal antibodies (mAbs) or the antibiotic polymyxin B (PMB) [9,158,159].…”

Section: Limitations Of Detection Assaysmentioning

confidence: 99%

“…The following factors were already reported to contribute to LER: temperature, pH, salinity, the presence of chelating agents (e.g., sodium citrate and EDTA), surfactants (e.g., Tween-20, Triton X-100, and polysorbate 20 and 80), and cationic proteins (e.g., lysozyme, ribonuclease A, and human IgG). These factors relate to both the sample and the buffers used during sample preparation or analysis [90,91,148,150,151,154,166,168,169]. Those factors can separate LPS from micellar into a less active monomeric form [148,150,167], or even interact directly with LPS, reducing its activity and solubility [122,170].…”

Section: Low Endotoxin Recovery and Endotoxin Potencymentioning

confidence: 99%

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Heterogeneity of Lipopolysaccharide as Source of Variability in Bioassays and LPS-Binding Proteins as Remedy

Fux

Melo

Michelini

et al. 2023

IJMS

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Lipopolysaccharide (LPS), also referred to as endotoxin, is the major component of Gram-negative bacteria’s outer cell wall. It is one of the main types of pathogen-associated molecular patterns (PAMPs) that are known to elicit severe immune reactions in the event of a pathogen trespassing the epithelial barrier and reaching the bloodstream. Associated symptoms include fever and septic shock, which in severe cases, might even lead to death. Thus, the detection of LPS in medical devices and injectable pharmaceuticals is of utmost importance. However, the term LPS does not describe one single molecule but a diverse class of molecules sharing one common feature: their characteristic chemical structure. Each bacterial species has its own pool of LPS molecules varying in their chemical composition and enabling the aggregation into different supramolecular structures upon release from the bacterial cell wall. As this heterogeneity has consequences for bioassays, we aim to examine the great variability of LPS molecules and their potential to form various supramolecular structures. Furthermore, we describe current LPS quantification methods and the LPS-dependent inflammatory pathway and show how LPS heterogeneity can affect them. With the intent of overcoming these challenges and moving towards a universal approach for targeting LPS, we review current studies concerning LPS-specific binders. Finally, we give perspectives for LPS research and the use of LPS-binding molecules.

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Section: Limitations Of Detection Assaysmentioning

confidence: 99%

Section: Limitations Of Detection Assaysmentioning

confidence: 99%

Section: Low Endotoxin Recovery and Endotoxin Potencymentioning

confidence: 99%

See 1 more Smart Citation

Heterogeneity of Lipopolysaccharide as Source of Variability in Bioassays and LPS-Binding Proteins as Remedy

Fux

Melo

Michelini

et al. 2023

IJMS

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“…В соответствии с нормативной документацией на вакцину брюшнотифозную Ви-полисахаридную определение содержания в ней пирогенных примесей проводится биологическим тестом на пирогенность 4 .…”

Section: Introductionunclassified

“…Согласно требованиям Евразийской экономической комиссии («Руководство по составлению нормативного документа по качеству лекарственного препарата») 5 определение бактериальных эндотоксинов (БЭ) с помощью ЛАЛ-теста является основным тестом для испытания парентеральных лекарственных препаратов. К преимуществам ЛАЛ-теста относят возможность проведения полуколичественного или количественного определения содержания БЭ [4], высокую чувствительность, хорошую воспроизводимость результатов 6 . Кроме того, метод отличает простота выполнения, возможность анализировать значительное количество образцов за короткий промежуток времени и экономичность [1].…”

Section: Introductionunclassified

Experimental evaluation of the possibility of determining bacterial endotoxins in typhoid Vi polysaccharide vaccines using the gel-clot test

Abramtseva,

Alekhina,

Kolyshkina

2023

Biological Products. Prevention, Diagnosis, Treatment

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Scientific relevance. Currently, only the rabbit pyrogen test is used to test the Vianvac® typhoid Vi polysaccharide vaccine for pyrogenicity. As part of the product specification file, the gel-clot test for bacterial endotoxins (BE) will improve the reliability of quality control, as well as harmonise the requirements for the vaccine with the requirements outlined for this group of medicinal products by leading world pharmacopoeias.Aim. This study aimed at an experimental assessment of the applicability of the gel-clot test to the quantification of BE in the typhoid Vi polysaccharide vaccine.Materials and methods. This study used samples from 5 batches of the typhoid Vi polysaccharide vaccine (0.5 mL/dose, solution for subcutaneous injection), LAL and TAL reagents. The analysis included the gel-clot test and the pyrogenicity test according to the State Pharmacopoeia of the Russian Federation (OFS.1.2.4.0006.15 and OFS.1.2.4.0005.15, respectively).Results. According to calculations, the BE limit for the tested vaccine was 96 EU/mL, and the maximum valid dilution (MVD) was 3200. The authors determined the regulatory requirements for typhoid Vi polysaccharide vaccine quality in terms of BE (not more than 48 EU/dose). The in vitro BE tests were positive at vaccine dilutions of 1/16 to 1/32 and negative at 1/64 to 1/256. The authors selected and validated a working vaccine dilution of 1/128. The BE content measured in the tested samples ranged from 0.24 to 0.48 EU/dose. The in vivo pyrogen tests were positive at dilutions of 1/16 to 1/128 and negative at 1/256 in all experiments with samples from 5 vaccine batches at dilutions ranging from 1/16 to 1/256.Conclusions. This study has experimentally proven that the gel-clot test can quantify BE in the typhoid Vi polysaccharide vaccine. The authors have recommended introducing the gel-clot BE test in the monograph of the State Pharmacopoeia of the Russian Federation on the typhoid Vi polysaccharide vaccine (FS.3.3.1.0012.15).

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Low endotoxin recovery and its impact on endotoxin detection

2021

Self Cite

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Endotoxin exists on the outer membrane of Gram-negative bacteria and poses risks to human health by triggering a series of immune responses. Therefore, its accurate detection is essential. The Limulus amoebocyte lysate (LAL) test is the most pharmacopeia-recognized and popular technique for endotoxin detection. Despite its wide industry adoption, the low endotoxin recovery (LER) phenomenon can compromise the LAL test's reliability. This review summarizes the possible reasons attributing to the LER phenomenon from three different perspectives: the endotoxin standards used in hold time study, protein active pharmaceutical ingredients, and excipients. Potential mechanisms and strategies to mitigate the LER phenomenon are also discussed as presented by different research groups.

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Effect of different sample treatment methods on Low Endotoxin Recovery Phenomenon

Cited by 6 publications

References 16 publications

Heterogeneity of Lipopolysaccharide as Source of Variability in Bioassays and LPS-Binding Proteins as Remedy

Heterogeneity of Lipopolysaccharide as Source of Variability in Bioassays and LPS-Binding Proteins as Remedy

Experimental evaluation of the possibility of determining bacterial endotoxins in typhoid Vi polysaccharide vaccines using the gel-clot test

Low endotoxin recovery and its impact on endotoxin detection

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