Endotoxin recovery using limulus amebocyte lysate (LAL) assay

Bolden, Jay S.; Warburton, Rob E.; Phelan, Robert W.; Murphy, Marie; Smith, Kelly R.; Felippis, Michael R. De; Chen, Dayue

doi:10.1016/j.biologicals.2016.04.009

Cited by 35 publications

(11 citation statements)

References 17 publications

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“…Our studies showing the activation of HEK293 bearing TLR4 and the corresponding partial reduction when the product was treated with Polymixin B strongly suggests that the product contains trace levels of endotoxin despite the negative finding using the LAL test. The LAL test is broadly used for testing endotoxin in therapeutic products 43 , however several conditions are known to reduce endotoxin recovery including the presence of surfactants, deoxycholate, or other formulation components that may alter endotoxin structure 44 , 45 . As shown in Supplementary Figure 2 , the ability of the LAL assay to detect endotoxin in the rhIFNβ products appears to correlate inversely with the albumin content.…”

Section: Discussionmentioning

confidence: 99%

Cell based assay identifies TLR2 and TLR4 stimulating impurities in Interferon beta

Haile

Polumuri

Rao

et al. 2017

Sci Rep

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Immunogenicity can have devastating consequences on the safety and efficacy of therapeutic proteins. Therefore, evaluating and mitigating the risk of product immunogenicity is critical for the development these products. This study, showed that Betaseron and Extavia, which are reported to be more immunogenic among IFNβ products in clinical usage, contain residual innate immune response modulating impurities (IIRMIs) capable of activating NF-κB and induced expression of inflammatory mediators. These IIRMIs were undetectable in Rebif or Avonex. The stimulatory effect was attributed solely to IIRMIs because it was evident in murine cells lacking the interferon receptor (IFNAR). The IIRMIs in Betaseron and Extavia triggered NF-κB activation in HEK-293 cells bearing TLR2 and TLR4 in MyD88 dependent manner. Importantly, the IIRMIs in Betaseron induced up-regulation of IL-6, IL-1β, and ccl5 in the skin of IFNAR knock out mice following subcutaneous administration. This indicates that trace level IIRMIs in Betaseron could contribute to the higher immunogenicity rates seen in clinics. Together these data suggest that cell based assays can reveal subtle but clinically relevant differences in IIRMIs following manufacturing changes or between products with the same active ingredients but different manufacturing processes. Appreciating these differences may inform immunogenicity risk assessments.

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Section: Discussionmentioning

confidence: 99%

Cell based assay identifies TLR2 and TLR4 stimulating impurities in Interferon beta

Haile

Polumuri

Rao

et al. 2017

Sci Rep

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“…labeled "lysate sensitivity (ë)" is the conc. of ET required to make the lysate to clot under optimum test conditions [27,28].…”

Section: Limulus Amoebocyte Lysate (Lal) Testmentioning

confidence: 99%

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

Elkhateeb

Elkhatib

Aboulwafa

2019

Archives of Pharmaceutical Sciences Ain Shams University

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End toxin is heat-stable lip polysaccharide (LPS) present in the outer membrane of the cell wall of Gramnegative bacteria. All parenteral preparations, as well as tissue implants, must be with no pyrogenic level of endotoxin or other related materials because of their associated health hazards and serious clinical effects. Accordingly, detection and limiting endotoxin in various pharmaceutical and biological products represent crucial issues. Rabbit pyrogen test (RPT) and Limulus Amebocyte Lysate (LAL) test are two methods used for endotoxin detection and quantification. Endotoxin detection is one of the most critical quality control tests required by the Food and Drug Administration (FDA) for all parenteral drugs in their final stage. Both in vitro LAL test and in vivo RPT can complement and reinforce each other but in certain cases, they are not interchangeable and they together provide a comprehensive picture of any potential contamination whether by endotoxin or any other pyrogenic matters.

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“…At present, the rabbit pyrogen test , and limulus amoebocyte lysate (LAL) − assays are the most common methods for the detection of bacterial endotoxin. Among them, LAL has the advantages of rapidity, simplicity, reliability, and high sensitivity (0.01–1 ng/mL), which has become the golden standard for endotoxin detection.…”

Section: Introductionmentioning

confidence: 99%

In Situ Detection of Endotoxin in Bacteriostatic Process by SERS Chip Integrated Array Microchambers within Bioscaffold Nanostructures and SERS Tags

Xiang

et al. 2020

ACS Appl. Mater. Interfaces

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In order to achieve real-time and in situ detection of endotoxin, which is an important and significant clinical test index, surface-enhanced Raman spectroscopy (SERS) chip integrated array microchambers within bioscaffold nanostructures and a SERS monitoring strategy were proposed in this paper. After sputtering of nanogold on the cicada wing, which was selected as a natural template, and polydimethylsiloxane bonding, array-type chambers within bioscaffold nanostructures were prepared for in situ bacterial culture and monitoring of endotoxin in the bacteriostasis process by SERS. Meanwhile, the SERS tag modified with the DNA aptamer was prepared and added into this complex biochemical reaction to further improve the sensitivity and selectivity. A new method for in situ detection of endotoxin was thus established. The detection time was shortened to 100 s, and the detection limit was as low as 6.25 ng/mL. Pseudomonas aeruginosa was cultured in situ in the chamber of the SERS chip with antimicrobial agents in 0−72 h. The endotoxin released in the antibacterial process was monitored by the designed SERS detection strategy. The results obtained by SERS analysis were consistent with those of the ELISA kit.

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Endotoxin recovery using limulus amebocyte lysate (LAL) assay

Cited by 35 publications

References 17 publications

Cell based assay identifies TLR2 and TLR4 stimulating impurities in Interferon beta

Cell based assay identifies TLR2 and TLR4 stimulating impurities in Interferon beta

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

In Situ Detection of Endotoxin in Bacteriostatic Process by SERS Chip Integrated Array Microchambers within Bioscaffold Nanostructures and SERS Tags

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