Recombinant Factor C

Bolden, Jay S.

doi:10.1007/978-3-030-17148-3_13

Cited by 5 publications

(5 citation statements)

References 12 publications

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“…Controlled-environment aquaculture and hemolymph collection could therefore alter the practice of wild harvest, expand the global LAL supply, and avert related viability threats, while building an irrefutable case for species conservation. Innovations like recombinant Factor C (rFC), an alternative to LAL assays, have emerged to address declining HSC populations (Bolden, 2019), with recent FDA approval for use of rFC in Eli Lilly's Emgality TM (galcanezumab). However, additional capital resources for equipment and end-user validation are also required to adopt such alternatives, which would be anticipated as significant drivers of industry demand and rapid acceptance of assays using abundant, sustainable, aquaculture-derived LAL.…”

Section: Resultsmentioning

confidence: 99%

Horseshoe Crab Aquaculture as a Sustainable Endotoxin Testing Source

et al. 2020

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Horseshoe crab (HSC) hemolymph is the source of Limulus amebocyte lysate (LAL), a critical component in sterility testing that ensures drug and medical device safety for millions of patients every year. Wild HSC populations have been declining as a result of its use as whelk and eel bait, environmental changes, and its capture and bleeding for hemolymph by the biomedical industry, thus posing significant risks to species viability and the LAL raw material supply chain. We designed a controlled aquaculture habitat to husband HSCs and evaluated the effects of captivity on health markers (e.g., amebocyte density, hemocyanin levels, and LAL activity). We found HSC aquaculture to be practicable, with routine hemolymph harvesting resulting in high LAL quality, while safeguarding animal well-being with 100% HSC survival. Further, lowimpact hemolymph harvesting via an indwelling catheter revealed rapid amebocyte rebound kinetics after consecutive 10% hemolymph extractions. Sustainable supplies of LAL could also be adapted to address daunting trends in septicemia and antimicrobial resistance. LAL is uniquely sensitive and specific for gram-negative bacteria, which represent 70-80% of pathogens that typically lead to sepsis. However, erratic results associated with interfering substances plagued efforts to adapt LAL for clinical use in the past. We report the development of a new LAL-based assay that can detect gram-negative bacteria and endotoxins in human blood without interference using aquaculture-derived LAL. Based on this research, sustainable LAL production from aquaculture could potentially satisfy industry needs with a fraction of one year's current capture via year-round harvesting from a finite cohort of HSCs and expand raw materials supplies for potential future clinical applications.

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Section: Resultsmentioning

confidence: 99%

Horseshoe Crab Aquaculture as a Sustainable Endotoxin Testing Source

et al. 2020

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“…However, as Bolden (2019 , p. 504) summarizes, it is ‘unproductive to pit the bait fishery take against the biomedical take.’ Indeed, as Davies (2019) has explored in the context of laboratory animals more broadly, drawing comparisons to the high numbers of animals used in other sectors does not reassure nor create a strong basis from which to argue an ethical point. However, interviewees felt that engaging with the specific numbers of crabs used biomedically did offer a route to creating a localized culture of care and interest in the 3Rs within endotoxin testing:…”

Section: Perspectives On Reducing Biomedical Horseshoe Crab Usementioning

confidence: 99%

“…Replacement refers to replacing animals with non-animal methods wherever feasible. A synthetic substitute to horseshoe crab blood was introduced in 2001 – laboratory-synthesized genetically engineered recombinant factor C (rFC) ( Ding and Ho, 2001 ), becoming commercially available in 2003 ( Bolden, 2019 ). Factor C here being ‘the endotoxin-sensitive serine protease that initiates the coagulation cascade’ in horseshoe crab blood ( Ding and Ho, 2001 , p. 277).…”

Section: Perspectives On Replacing Biomedical Horseshoe Crab Usementioning

confidence: 99%

Atlantic Horseshoe Crabs and Endotoxin Testing: Perspectives on Alternatives, Sustainable Methods, and the 3Rs (Replacement, Reduction, and Refinement)

Gorman

2020

Front. Mar. Sci.

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Endotoxin testing is a vital part of quality and safety control in pharmaceutical production. The primary method for this testing in North America and Europe is the limulus amebocyte lysate (LAL) test, a critical component of which is the blood of Atlantic horseshoe crabs (Limulus polyphemus). Procuring blood for LAL testing involves capturing and bleeding over 500,000 crabs from wild marine populations each year. Whilst efforts are made by manufacturers to return crabs to the sea following the collection of blood, there is a level of mortality and sub-lethal impact involved, prompting increasing discussions about welfare and ethics. The 3Rs-the ambition to where possible, replace, reduce, and refine the use of animals-are established and accepted worldwide as the best framework for governing animal-dependent science. However, the biomedical utilization of horseshoe crabs to produce the LAL test has rarely been viewed through a 3Rs framework. More recently, there has been a renewed attention on sustainable methods and alternatives to the LAL test. Drawing on in-depth qualitative interviews, this article examines stakeholder perspectives on opportunities for thinking with the 3Rs, considering current appetites to replace, refine, and reduce contemporary biomedical reliance on horseshoe crabs. The shape of conversations about the biomedical utilization of horseshoe crabs has shifted significantly in recent years, and the 3Rs are an important driver of change, offering the potential to advance the use of more sustainable methods, and realize the welfare considerations increasingly expected across science and society.

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“…rFC is a genetically engineered protein, which is activated by endotoxins to produce a fluorescent end product which is quantifiable. 12 The RPT method involves measuring the possible rise in temperature of three rabbits following the intravenous injection of a test solution in each rabbit. 13 In the final method, the MAT, a sample is incubated with human monocytes which produce cytokines/IL if pyrogens are present, that are detected in an ELISA.…”

Section: Introductionmentioning

confidence: 99%

The monocyte activation test detects potentiated cytokine release resulting from the synergistic effect of endotoxin and non-endotoxin pyrogens

Solati¹,

Zhang²,

Timman³

2022

Innate Immun

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Pyrogens are classified in two groups, endotoxin pyrogens and non-endotoxin pyrogens (NEPs). The presence of either in parenteral pharmaceuticals or medical devices can cause severe harm to subjects, and when occurring in combination, synergistic potentiation effects can occur. As the standard in vitro pyrogen test, the Limulus Amebocyte Lysate (LAL) assay can detect LPS only, an endotoxin, but not NEPs. We tested whether the Monocyte Activation Test (MAT) that measures IL-6 induction, is suited for detecting synergistic pyrogen effects. Here we show that MAT reliably detects the NEPs heat-killed Staphylococcus aureus, R848 and lipoteichoic acid, in addition to LPS. When combinations of these pyrogens were tested, a potentiation of IL-6 production was seen beyond an additive effect, apparently reflecting on in-vivo synergisms. The current study therefore demonstrates that MAT not only is a reliable and reproducible assay for the sensitive detection of both endotoxin and non-endotoxin pyrogens, but also for identifying synergistic effects when parenteral drugs are contaminated with multiple pyrogens.

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Recombinant Factor C

Cited by 5 publications

References 12 publications

Horseshoe Crab Aquaculture as a Sustainable Endotoxin Testing Source

Horseshoe Crab Aquaculture as a Sustainable Endotoxin Testing Source

Atlantic Horseshoe Crabs and Endotoxin Testing: Perspectives on Alternatives, Sustainable Methods, and the 3Rs (Replacement, Reduction, and Refinement)

The monocyte activation test detects potentiated cytokine release resulting from the synergistic effect of endotoxin and non-endotoxin pyrogens

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