Currently Available Recombinant Alternatives to Horseshoe Crab Blood Lysates: Are They Comparable for the Detection of Environmental Bacterial Endotoxins? A Review

Bolden, Jay S.; Knutsen, Chris; Levin, Jack; Milne, Catherine T.; Morris, Tina S.; Mozier, Ned M.; Spreitzer, Ingo; Wintzingerode, Friedrich von

doi:10.5731/pdajpst.2020.012187

Cited by 8 publications

(7 citation statements)

References 13 publications

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“…A fundamental difference between rFC and LAL could not be shown and therefore demonstrated equivalent specificity. A metastudy on rFC vs. LAL comparisons confirmed these results (13).…”

Section: Do We Need To Demonstrate All 8 Aspects?supporting

confidence: 57%

Recombinant Factor C Validation—Simpler Than You Think!

Uhlig¹,

Williams²,

Tindall³

2021

PDA Journal of Pharmaceutical Science and Technology

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Some users have been hesitant to take on the validation of alternative methods. However, recombinant factor C (rFC) tests are so highly analogous to the horseshoe crab-sourced Limulus amebocyte lysate (LAL) tests that users should consider their advantages. They include sustainability, safeguarding the supply chain due to geographical diversity, accuracy, specificity, and reproducibility. Also noteworthy is that rFC is gaining acceptance in most of the world, with its own compendial chapter in the European pharmacopoeia (2.6.32)(1) and a new section written in the Chinese Pharmacopeia. Although given the delay of the USP, let us consider the "ease of validation" according to USP <1225> (2) as detailed in Q&A format below.What needs to be done for rFC Method Validation? With an operationally qualified reader installed, generally a full validation can be achieved in as little as 2 days, with 6 rFC assays, incl. 2 operators and 3 reagent lots to determine all necessary validation parameters.

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“…A fundamental difference between rFC and LAL could not be shown and therefore demonstrated equivalent specificity. A metastudy on rFC vs. LAL comparisons confirmed these results (13).…”

Section: Do We Need To Demonstrate All 8 Aspects?supporting

confidence: 57%

Recombinant Factor C Validation—Simpler Than You Think!

Uhlig¹,

Williams²,

Tindall³

2021

PDA Journal of Pharmaceutical Science and Technology

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“…Recombinant alternatives are specific to endotoxin, and consist of two types of reagents: recombinant Factor C and cascade enzymes (see “next-generation LAL technology”). Recently, Bolden et al reviewed currently available recombinant alternatives to horseshoe crab blood lysates and their comparability [ 45 ].…”

Section: Various Techniques Involving the Lal Assay And Other Methodsmentioning

confidence: 99%

“…The LAL assay has established a firm position as an alternative to the rabbit pyrogen test, and thus, the horseshoe crab has already proven to be an extremely beneficial organism for biomedical use. However, there is growing awareness of the importance of protecting endangered species, and thus, alternative assay technologies using recombinant LAL have gained attention with data accumulation [ 45 , 83 ]. As stated earlier, clinical evaluation of the LAL assay remains challenging for a number of reasons, including various interferents and the emerging issue of endotoxin masking.…”

Section: Future Perspectivementioning

confidence: 99%

Outstanding Contributions of LAL Technology to Pharmaceutical and Medical Science: Review of Methods, Progress, Challenges, and Future Perspectives in Early Detection and Management of Bacterial Infections and Invasive Fungal Diseases

Tamura

Reich²,

Nagaoka

2021

Biomedicines

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The blue blood of the horseshoe crab is a natural, irreplaceable, and precious resource that is highly valued by the biomedical industry. The Limulus amebocyte lysate (LAL) obtained from horseshoe crab blood cells functions as a surprisingly sophisticated sensing system that allows for the extremely sensitive detection of bacterial and fungal cell-wall components. Notably, LAL tests have markedly contributed to the quality control of pharmaceutical drugs and medical devices as successful alternatives to the rabbit pyrogen test. Furthermore, LAL-based endotoxin and (1→3)-β-D-glucan (β-glucan) assay techniques are expected to have optimal use as effective biomarkers, serving as adjuncts in the diagnosis of bacterial sepsis and fungal infections. The innovative β-glucan assay has substantially contributed to the early diagnosis and management of invasive fungal diseases; however, the clinical significance of the endotoxin assay remains unclear and is challenging to elucidate. Many obstacles need to be overcome to enhance the analytical sensitivity and clinical performance of the LAL assay in detecting circulating levels of endotoxin in human blood. Additionally, there are complex interactions between endotoxin molecules and blood components that are attributable to the unique physicochemical properties of lipopolysaccharide (LPS). In this regard, while exploring the potential of new LPS-sensing technologies, a novel platform for the ultrasensitive detection of blood endotoxin will enable a reappraisal of the LAL assay for the highly sensitive and reliable detection of endotoxemia.

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“…In August 2020 the PDA Journal of Pharmaceutical Science and Technology published a review of currently available recombinant alternatives to HSC blood lysates [65]. This analysis was completed by members of the PDA Endotoxin Leadership Forum, which included members of four pharmaceutical manufacturers, three members of scientific organizations and Jack Levin, one of the original inventors of the LAL assay [9].…”

Section: Latest Reviewmentioning

confidence: 99%

Recombinant Bacterial Endotoxin Testing: A Proven Solution

Tindall

Demircioglu²,

Uhlig³

2021

BioTechniques

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Testing of parenteral pharmaceuticals and medical devices for pyrogens (fever-inducing substances) is critical to patient safety. The original rabbit pyrogen test has largely been replaced by different bacterial endotoxin tests based on Limulus amebocyte lysate (LAL), sourced from the blood equivalent of horseshoe crabs after comparative studies to the rabbit pyrogen test. Since 2004 a bacterial endotoxin test based on recombinant factor C (rFC), the endotoxin sensor protein inside of LAL, has been used as an animal-free alternative to LAL. Likewise, numerous studies compared LAL and rFC. Here we describe the history of pyrogen and bacterial endotoxin testing and summarize the evidence presented by those studies. We demonstrate that rFC and LAL are equivalent and comparable.

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Currently Available Recombinant Alternatives to Horseshoe Crab Blood Lysates: Are They Comparable for the Detection of Environmental Bacterial Endotoxins? A Review

Cited by 8 publications

References 13 publications

Recombinant Factor C Validation—Simpler Than You Think!

Recombinant Factor C Validation—Simpler Than You Think!

Outstanding Contributions of LAL Technology to Pharmaceutical and Medical Science: Review of Methods, Progress, Challenges, and Future Perspectives in Early Detection and Management of Bacterial Infections and Invasive Fungal Diseases

Recombinant Bacterial Endotoxin Testing: A Proven Solution

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