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Figure 1. The four pillars supporting rFC adoption. (1) Established
utilitiy of LAL, (2) Biotechnological replacement of natural
proteins using recombinant proteins, (3) Understanding
resistance /Overcoming roadblocks and (4) Growing acceptance.

(a) The horseshoe crab harbors a plethora of formidable innate
immune defense molecules and mechanisms, which has protected
this "living fossil" for over 450 million years from extremely challenging
microbes. In 1956 Frederick Bang discovered blood clotting in Limulus
when infected by GNB.1 Levin and Bang revealed the overlap in
blood clotting mechanisms, demonstrating evolutionarily conserved
elements in human thrombin and Limulus Factor C.2-3 The clotting of
Limulus hemolymph was correlated with the development of fever in
rabbits and humans. LAL was thus developed and approved by FDA
in 1977 as an alternative to RPT for endotoxin test and it was USPregistered in 1983. Biochemists further characterized the protein
constituents in the coagulation cascade,4-14 and showed a series of
serine proteases (Factor C, Factor B, Factor G and proclotting enzyme)
in the blood cells, and amoebocytes, which is responsible for blood
coagulation in the presence of endotoxin (Figure 2). The simultaneous
establishment of a control standard endotoxin (CSE) and eventually
a reference standard endotoxin (RSE), allowed for the continued
correlation of clotting to fever- from endpoint test to kinetic tests. This
important body of scientific data serves as a platform to support the
utility of both LAL and rFC, and need not be re-established for rFC,
given the ongoing use of RSE and derived CSE.
The voluminous hemolymph in horseshoe crab has been deemed a
gift to humankind. Turning this gift into a fortune, LAL-producers have
marketed the LAL-based endotoxin test worldwide in various test
formats: gelation, turbidometric, endpoint and kinetic colorimetric/
fluorometric assays. The establishment of the utility of LAL by the
pharmaceutical industry's need for QC of parenterals for human use
has unfortunately led to resolute harvesting and over-harvesting of
horseshoe crab. For LAL production, horseshoe crabs are lined up for
"involuntary donations" of life-saving blood. The persistent glut for the
horseshoe crab blood has driven the species to near extinction.15-21
(b) The assumption of switching from RPT to LAL test includes the posit
that endotoxin is the only significant pyrogen of water-based drug
manufacturing. This seems a huge leap of faith, however the ubiquity
of GNB in water environments, the potency and stability of endotoxin
relative to other potential microbial contaminants made the case that
the real risk in traditional pharma manufacturing lie in the potential for
rapid growth of GNB in water systems and the generation of endotoxin

Figure 2. (a) Amoebocytes from horseshoe crab hemolymph.
There are two types of electron dense granules, the large
and small granules. Antimicrobial peptides are present in the
small granules while Factor C and other coagulation cascade
enzymes are present in the large granules. (b) The coagulation
cascade constituting serine proteases present in LAL (Limulus
amoebocyte lysate), TAL (Tachypleus amoebocyte lysate) and CAL
(Carcinoscorpius amoebocyte lysate). Factor C is the endotoxinsensitive enzyme, which triggers coagulation of LAL. The
presence of fungal contaminants, plant by-products or algae or
cellulosic filter leaches give rise to β-glucans which also trigger
coagulation through Factor G, causing false-negative results in
endotoxin test.

in this key drug constituent. The indomitable endotoxin can only be
removed or destroyed by ultrafiltration or extreme dry heat-treatment.
(c) Given the potential growth of GNB in even the purest of water,
the statistical coverage of testing the entire manufacturing process
rather than "spot checking" the end-product is perceived as the
real risk in drug manufacturing. By early and frequent in-process
testing, contamination risks could be precluded. However, this
widespread testing is unachievable with RPT because of the laborious,
uneconomical and unsustainable nature of rabbit colonies. More
importantly, the rapidity of endotoxin-test results required for inprocess monitoring is only feasible with in vitro LAL tests.

Pillar 2: Biotechnological
Development: Three to Four Decades
of Replacing Natural Proteins with
Recombinant Proteins Established a
Successful Paradigm
The supporting framework for Pillar 2 includes the: (a) development
of recombinant technologies that have allowed for the replacement
of natural animal-derived proteins and (b) fragility of the horseshoe
crab supply due to over-harvesting and environmental decline (Figure
3). The sustainability and supply chain of LAL has been taken for
granted.17 If risk control philosophy were re-examined, one would
expect to see more companies validating rFC as a backup test. The
regulations against over-harvesting of horseshoe crabs are daunted
by difficulties with tracing and record-keeping of the harvest. Since
2004, the Atlantic States Marine Fisheries Commission (ASMFC) started
documenting harvest-related mortality. In 2018, approximately half
a million horseshoe crabs were harvested and bled, with ASMFC's

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