APR September/October 2020 - 158

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MICROBIOLOGY

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Endotoxin Detection:
The Four Pillars of rFC Adoption in Lieu of LAL
Jeak Ling Ding,1+* Gregory Devulder,2+
Holger Grallert,3 Kevin Williams,2 and Bow Ho4
Department of Biological Sciences, National University of Singapore, Singapore
bioMérieux, 5 Rue des Aqueducs, 69290 Craponne, France
3
Hyglos GmbH, bioMérieux, Am Neuland 1/3, 82347 Bernried, Germany
4
Department of Food Science and Technology, National University of Singapore, Singapore
1
2

+

Co-senior authors
*Corresponding author: dbsdjl@nus.edu.sg
http://www.dbs.nus.edu.sg/staff/djl.htm

Abstract
Endotoxin from Gram Negative Bacteria (GNB) interacts with cellular
receptors to trigger strong systemic inflammatory responses
including fever, septic shock and death. The threat of endotoxin to
the pharmaceutical and healthcare industries cannot be overstated.
Unless GNB and endotoxin contamination in parenteral drugs and
implantable devices are rapidly and reliably detected to enable their
elimination, peril lurks. The quality control (QC) of biomedical products
begun in 1942 with the slow, laborious and costly rabbit pyrogen test
(RPT). Then in 1977, the US FDA approved the Limulus amoebocyte
lysate (LAL) test. However, the LAL test requires harvesting and
bleeding of the endangered horseshoe crab, disrupting the ecosystem.
The emerging acceptance of recombinant Factor C (rFC) in lieu of LAL
rests upon specific pillars that include the advantages of rFC and also
stands upon the original established utility of LAL as a replacement
for RPT.
The first pillar of rFC adoption is the established utility of LAL which
first used the blood of an arthropod, Limulus polyphemus, in place of
RPT. It was then necessary to establish the relevance of an arthropod
immune system to the mammalian response as well as to demonstrate
that endotoxin is the predominant biological pyrogenic contaminant
of water-based drug manufacturing, rather than previous concerns of
detecting all pyrogens.
The second pillar of rFC includes three to four decades of
biotechnology efforts to develop recombinant technologies that
have allowed for the transformation of therapeutic capabilities,
including recombinant drugs, monoclonal antibodies, gene therapy,
and understanding disease causation at a molecular-genetics level.

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These efforts include developing an alternative, highly reproducible
synthetic endotoxin-biosensor, rFC. The second pillar also rests on the
fragility of harvesting and bleeding vulnerable endangered animals to
manufacture LAL.
The third pillar of rFC explores why rFC has not been more widely
adopted to date; what have been the roadblocks? We review man-made
reasons as there are no inadequacies associated with biotechnological
production of synthetic rFC; furthermore, the rFC single enzyme-based
endotoxin detection technology is widely accepted and has provided
a revolution in therapeutic safety and efficacy.
The fourth pillar of rFC elaborates the way forward for rFC in terms
of validation and compendial acceptance which is rapidly gaining
recognition towards utility worldwide. This pillar rests upon the
significant features of rFC, including standardized operating procedures
and production, which will allow for its widespread adoption.
The adoption of rFC to replace LAL, which had previously replaced
RPT, rests on four pillars supported by the benefits of the synthetic rFC
(Figure 1).

Pillar I: The Utility of LAL
The utility of LAL over the in vivo RPT depended upon three
demonstrable facets of human physiology and pharmaceutical
manufacturing, including: (a) the establishment of the relevance of an
arthropod immune system to human fever reactions, (b) the posit that
the only significant pyrogen of water-based drug manufacturing is
lipopolysaccharide or endotoxin from GNB, and (c) the ease of use and
applicability of the in vitro LAL test in expanding drug manufacturing
coverage from a statistical vantage.

| September/October 2020

10/2/20 11:46 AM
http://www.dbs.nus.edu.sg/staff/djl.htm
APR September/October 2020

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