Pr. J. L. Ding - A. Endotoxin is a type of pyrogen which causes fever. It is a component of the cell wall of Gram-negative bacteria (GNB), such as E. Coli. Endotoxin is naturally present on our skin, in our gut, in water and many other materials, however, when endotoxin enters the bloodstream, it induces inflammation and pyrogenic reaction (fever). If allowed to persist either through blood infection by GNB or as a contaminant introduced via injected medicines or implanted medical devices, the endotoxin can cause sepsis and even death. Accurate and reliable testing of parenteral pharmaceuticals and medical devices for the presence of endotoxins is therefore, a crucial quality control test to ensure their safety for human use. 

Prior to the introduction of rFC, two methods had commonly been used for endotoxin testing: the in vivo rabbit pyrogen test (RPT) and the “Limulus amoebocyte lysate” (LAL) test which uses blood harvested from horseshoe crabs. Both methods rely on the use of animals, and have other limitations and drawbacks.

The RPT can only provide qualitative results, has variable sensitivity to endotoxin and is prone to false results. While the LAL test is quantitative, it has batch-to-batch variations in sensitivity and specificity to endotoxin, and there is no one standardized methodology for processing assays. The presence of β glucan in test samples and other non-specific proteins in LAL can also interfere with test outcomes, increasing the potential for false positives or erroneously higher endotoxin-positive readouts.

On our journey to engineering a new method for endotoxin testing, we aimed to address these limitations and were also inspired to help overcome the existential threat to horseshoe crab survival; one of the main contributors to the increasing risk is the over-harvesting to produce LAL. 

Endotoxin activates Factor C, which triggers the Limulus blood coagulation cascade reaction that powers the LAL test. It follows that Factor C offers an ideal target upon which we could base a new method for endotoxin testing – by cloning Factor C we could produce a synthetic recombinant Factor C (rFC), which is stable, scalable, safe, sustainable and efficient. Having a genetically engineered source of rFC ensures an animal-free perpetual supply of material for endotoxin testing, ‘on-tap’ from the rFC clone. Expressed from the selected single and unique genetic clone, the rFC provides an invariable, highly sensitive and specific test for endotoxin, with high throughput capacity for quality assurance of the drug manufacturing process; from testing in-house water and raw materials to the pharmaceutical end-product.

The introduction of rFC has transformed the way we conduct endotoxin testing, making it more simple, accurate and robust. Whereas the LAL test involves a coagulation cascade of multiple enzymes, where each member switches on the next in the series, as a single enzyme-based assay the rFC alone is sufficient and invariable. This means that the quality control of the rFC-based endotoxin test kit itself is more straightforward compared to that of LAL-based test kits. The rFC test is also very easy to adopt, since the workflow for rFC-based test is very similar to that of LAL-based tests. Furthermore, most labs previously using LAL-based tests will already be equipped with the necessary facilities for the assay.

Thanks to the use of rFC, the endotoxin test is more efficient, standardized, integrated and cost-effective, ensuring the quality and safety of pharmaceuticals and medical devices for parenteral use. A growing body of evidence is also being established through studies regarding the valuable role of rFC in endotoxin testing, and this is also being increasingly recognized by regulators.

Encouragingly, in 2021, the European Pharmacopoeia accepted and approved rFC with chapter Ph. Eur. 2.6.32, enabling pharmaceutical manufacturers across 39 European countries to utilize it. In addition, the European Pharmacopoeia Commission further solidified rFC’s acceptance by approving revisions to include this test in several monographs. More recently, rFC testing was included in the British Pharmacopoeia published in January 2024, and in July 2024 the US Pharmacopoeia’s Microbiology Expert Committee voted in favor of including a new chapter <86> in the US Pharmacopoeia which endorses non-animal derived reagents for endotoxin testing, including rFC and recombinant cascade reagents (rCR). Chapter <86> provides manufacturers with information needed to use non-animal derived reagents for endotoxin testing. 

The complete value of rFC as a solution for endotoxin testing is a topic I will be covering at length in an upcoming webinar with bioMérieux.  

In the future, we could envisage rFC-based testing expanded to in situ detection of endotoxin or live GNBs, on solid materials. While the rCRs, which consist of three recombinant proteins in the LAL cascade, namely rFC, rFB and rProclotting enzyme, are being explored, it is crucial to consider the practical implications. From a supply chain perspective, rFC allows for simplification, reduction on environmental impact, and enhanced sustainability as fewer critical reagents need to be produced and managed. As a single enzyme, the rFC-test kit itself requires only a single quality control and ensures a more reliable and streamlined production process, making rFC a more efficient and sustainable choice for endotoxin testing.  Additionally, we may see the emergence of different new formats and platforms for processing rFC and rCR assays. 

It is very encouraging to see more leading companies in the field of microbiological testing taking an active role in supporting wider uptake of more sustainable, ethical forms of endotoxin testing. One such company has been bioMérieux, who are experts in industrial microbiology testing and have been incredibly committed to relying on endotoxin testing that uses rFC alone, without parallel harvesting of horseshoe crabs or buying blood for LAL production. This focus has set bioMérieux apart as they continue to develop creative and innovative methods of endotoxin detection with rFC. It also speaks to their credentials in sustainability along with their commitment to ethical practices. 

In the Asian-Pacific region, horseshoe crabs are now considered endangered and the impact of human activities on their habitat has led some species to disappear completely in certain areas. The knock-on effects of diminishing horseshoe crab populations are also concerning, with other species in the ecosystem being reliant on their eggs as a source of food. If we have a solution that can avoid putting these species in danger and reduce our environmental impact, surely it is our duty to put this into practice?

Furthermore, from a practical viewpoint, by relying on LAL as their material of choice for endotoxin testing, companies leave themselves at risk from the consequences of any disruption to their supply – for example if a natural disaster were to occur and impact the fragile habitat causing the loss of the remaining horseshoe crab population, this could disrupt whole supply chains.

Over 50 years ago the 3Rs principle was created as an ethical framework for performing animal research, encouraging the replacement, reduction, and refinement of tests that rely on animals. The development of rFC is a genuine reflection of these principles in action and has unlocked the possibility of eliminating our reliance on animal-based methods for endotoxin testing. We remain optimistic that the tide is turning thanks to greater recognition of recombinant proteins as the endotoxin testing method of choice. With greater uptake of rFC, endotoxin testing can become fully sustainable and ethical, helping to conserve vulnerable and endangered species and support the future of our planet.