Endotoxin Detection: The Four Pillars of rFC Adoption in Lieu of LAL

Authors:

Jeak Ling Ding,^1+* Gregory Devulder,²⁺
Holger Grallert,³ Kevin Williams,² and Bow Ho^4
1Department of Biological Sciences, National University of Singapore, Singapore
²bioMérieux, 5 Rue des Aqueducs, 69290 Craponne, France
³Hyglos GmbH, bioMérieux, Am Neuland 1/3, 82347 Bernried, Germany
⁴Department of Food Science and Technology, National University of Singapore, Singapore

+Co-senior authors
*Corresponding author: [email protected]
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. 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.

(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 scientifi c 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 in this key drug constituent. The indomitable endotoxin can only be removed or destroyed by ultrafi ltration 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 estimated mortality of 75,000 after being released into the wild.¹⁵ The decline in the horseshoe crab population has disrupted the ecosystem, with knock-on effects on the survival of migratory birds which depend on the horseshoe crab eggs for energy to complete its trans-hemispheric flight.¹⁶

The potential of rFC to substitute for LAL has taken ~40 years of recombinant protein development to become well-established. The fi rst commercial recombinant human insulin, Humulin^® was manufactured by Eli Lilly in 1982. Thenceforth, dozens of life-saving molecules e.g. growth factors, blood factors, cytokines, replacement enzymes, monoclonal antibodies, hormones and vaccines have emerged. If the structure of a recombinant molecule is identical to a natural protein, the function has been shown to be directly equivalent. Current contentions that rFC is non-equivalent to naturally harvested blood lysates should, but often does not, point directly to the false positive recoveries via LAL, of non-endotoxin constituents in various samples. The Factor G pathway contained in LAL22 raises the recovery values in natural waters which essentially contain all of nature including β-glucans and cellulosic residues from wood and grass. Roslansky and Novitsky²³ showed that β-glucans (laminarin, curdlan and cellulose residues) react differently to different batches of LAL depending on whether the LAL preparation used chloroform or, if it contains zwittergent. Thus different formulations of LAL produce different results when samples are contaminated with β-glucans. The expectation that “rFC versus LAL” tests will always agree when testing uncharacterized samples, whereas “LAL versus LAL” tests do not agree, is therefore unreasonable.

For decades, molecular biotechnologists have attempted to turn the tide to save the horseshoe crab and produce a sustainable, environmentally-friendly and eco-friendly synthetic endotoxin biosensor. Based on accumulated knowledge on the coagulation cascade enzymes, the primer serine protease Factor C, which is extremely sensitive to endotoxin, was genetically-engineered and cloned.^24-33 It took 15 years- from gaining a full understanding of the biochemical nature of Factor C; to cloning using semi-traditional molecular biology techniques; to expressing fully functional recombinant Factor C protein in various hosts (bacteria, yeasts, insect cells, mammalian cells). The recombinant Factor C (rFC) was finally coaxed out of its web of multi-functional domains into an enzymatically competent precursor ready to unleash its catalytic activity when provoked by endotoxin.^26,30 The rFC offers a neat, simple and single-enzyme test which is specific for bacterial endotoxin. It only requires a colorimetric or fluorogenic substrate to produce a color or fluorescence readout, to measure endotoxin (Figure 4).

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The rFC acts independently and does not suffer interference from other LAL proteins. Yet, it has taken another 20 years: from outlicensing the rFC clone; to waiting for the FDA and regulatory approvals and Pharmacopoeial endorsements; to contending with the established utility of conventional LAL. Meanwhile, the horseshoe crabs dwindled to near extinction. But, rFC perfectly fits the 3Rs: Refinement, Reduction, Replacement; a 4th R, Reproducibility of endotoxin detection, befits the rFC. The advantages of adopting rFC to replace LAL are obvious.

Pillar 3: Why the Resistance and Roadblocks to Embrace rFC Despite its Proven Efficacy?

In the biotechnology-biomedical sector, absolutely high-grade research biologics and diagnostics are required for healthcare. For the highly regulated drug industry, compendial status is imperative to avoid regulatory scrutiny of end-products. The adoption of a reliable alternative assay such as rFC for water, raw material, and inprocess testing is crucial, and it will replace up to 90% of LAL usage. However, the pharmaceutical companies and healthcare industries have relied largely on LAL for nearly half a century, despite problems associated with the LAL assay. The first rFC test for endotoxin has been commercially available since 2004 and most recently in 2011.

However, recognition of the rFC test was hampered by the continuing production of LAL and the intractable usage of LAL test. The rFC is a tried, tested and reliable replacement for LAL. The rFC is readily amenable to high throughput and automation in various formats, which benefits pharmaceutical companies. The advantages associated with rFC have been referred to as the “3 S’s” which include (a) scientific characterization, (b) sensitivity and (c) specificity.

Pillar 4: The Way Forward for rFC Adoption - Regulatory and Pharmacopoeial Acceptance of rFC (US, EU, China)

For many years, scientists have envisioned that research findings on the horseshoe crab innate immunity will benefit the biotechnology, pharmaceutical and human healthcare industries. Factor C and many other evolutionarily conserved molecules have been identified as having utility in immune defense, antimicrobial action and treating sepsis.^34-44 The use of LAL should therefore be limited to research into potentially life-saving and life-enhancing molecules. The applications of the rFC-based endotoxin detection have been supported worldwide by research applications; it has also eased the demand for LAL in cleanroom technologies.⁴⁵ The use of rFC by Eli Lilly Inc.⁴⁶ for QC of drugs, four of which have been successfully endorsed by US FDA, is testament to the reliability and worthiness of rFC for validation of parenteral drugs. The pharma companies must no longer tolerate the inertia to adopt rFC. The continuing harvest of the Limulus is not prudent. In fact, it risks the entire supply chain of pharmaceutical products. When the last ^Limulus is bled and there is no more LAL, what options will the disparagers of rFC have? Recent efforts to promote the use of synthetic rFC in place of LAL, to conserve the horseshoe crab from extinction,^15-21 have accelerated the adoption of rFC as the compendial test. A growing list of rFC regulatory acceptance has accumulated (Table 1).

The much anticipated COVID-19 vaccine under production by >100 companies will benefit with the utility of rFC for efficient in-process production and quality assurance.

Acknowledgements

This work was supported by The Ministry of Education, The National Medical Research Council, The Biomedical Research Council, The National University of Singapore, Singapore and bioMerieux Inc., an in vitro diagnostic company.

Author Biographies

Jeak Ling received her PhD from University of London, UK; in the area of Biochemistry and Cell Biology. After postdoctoral trainings in London and Singapore in innate immunology and cancer biology, she joined the National University of Singapore. Research, education and graduate administration are her main focus. Her research interest in host-pathogen interaction, in collaboration with Bow Ho (microbiologist), led to cloning and expression of synthetic rFC from the horseshoe crab, which constitutes the Pyrogene® test for endotoxin. More information at: http://www.dbs.nus.edu.sg/staff/djl.htm

Gregory Devulder studied Biological and Biosystems Engineering at the Universities of Nantes and obtained his PhD at the University of Lyon, France, in 2004. He has had different R&D positions of increasing responsibility at bioMérieux, in both Food and BioPharma divisions in France and USA. Currently, he is the Endotoxin Program Director, and shares his time between France and Germany. In his role, Gregory is responsible for the bioMérieux development program of endotoxin testing solutions.

Holger Grallert studied Biology and received his PhD degree at the Technical University of Munich (TUM). For several years, he was the head of the R&D unit of Hyglos GmbH and is now the manager of the R&D unit “Process and Product Impurities” of BioMérieux Industry with localization in Bernried, Germany. His unit is focused on the improvement of existing and development of new analytical methods in the field of Pyrogens and bacterial Endotoxins.

Kevin Williams spent 30 years at Eli Lilly & Company developing endotoxin assays and detection technology in the QC lab. He then worked at Hospira (now Pfizer), Lonza, GE water before moving to bioMerieux. He has authored several books on endotoxin (Endotoxin second and third edition, informa) including, most recently, Endotoxin Detection and Control in Pharma, Limulus and Mammalian Systems (2019, Springer Nature). He is frequent speaker at endotoxin related industry events.

Bow Ho received his PhD from University of Wales, UK; in the area of Microbiology. After industry experience at Boots Pharmaceutical, UK, he joined the Microbiology Dept, National University of Singapore (NUS). He is currently an adjunct faculty at the Food Science & Technology, NUS. His expertise in the area of rapid microbiology methodology led to the initial interest in developing a Gram negative bacterial endotoxin test with the hemolymph of Carcinoscorpius rotundicauda. Integrating knowledge in microbiology and in collaboration with Jeak Ling Ding, the synthetic rFC was innovated.

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