Bacterial endotoxin testing occurs at all stages of sterile pharma manufacturing, from raw materials to semifinished and final product. This ensures safe production of injectables and medical devices. Mandated by regulators, endotoxin control is important for two main reasons: First, endotoxins are known to cause strong human immune-system reactions, including fever, and can proceed to Gram-negative bacterial sepsis. Second, even trace amounts of endotoxin may interfere with the immune response of medical drugs, potentially altering their therapeutic effects.
Limulus amoebocyte lysate (LAL) and tachypleus amoebocyte lysate (TAL), obtained from Atlantic and Asian horseshoe crabs respectively, have been used to detect endotoxin for decades. More recently, the pressure on conservation of horseshoe crabs has increased due to dwindling populations and related ecological consequences, e.g., drastic declines of migrating birds. An article in the January 2017 issue of Nature states: "The main problem for the rufa red knots is thought to lie more than 3,000 kilometres to the south. During their migration from South America, the birds stop to feed on energy-rich eggs laid by horseshoe crabs (Limulus polyphemus) in Delaware Bay. Research suggests that the crabs have been so overharvested that the red knots have become deprived of much-needed fuel" (3).
Vulnerable horseshoe crab populations in North America and critically low numbers in Asia will make it difficult to meet the growing need from the pharmaceutical industry (4). But modern recombinant DNA technology has been well-established for safe, affordable and sufficient production of reagent proteins to replace the animal source. The molecular mechanism of the natural LAL/TAL reaction was first described in detail in the 1980s (5). Central to this mechanism is the zymogen Factor C that functions as a biosensor responding to endotoxin. This essential role of Factor C was the key reason that Dr Lynne Ding chose to express recombinant Factor C (rFC) as an alternative to LAL/TAL (6). Since then, further efficient and controlled rFC production processes have been established. The inherent advantages of recombinant production are that the reagents are biologically and chemically defined, as well as assured of high purity and lot-to-lot consistency. The known drawbacks associated with natural amoebocyte lysates, such as variation due to seasonal fluctuations of the animal-derived material, are thereby overcome (7).
In rFC endotoxin tests, following activation by endotoxin present in a sample, rFC cleaves a substrate which, in turn, emits a quantifiable fluorescence signal. All available rFC assays follow conventional bacterial endotoxin testing (BET) methodology in terms of acceptance criteria, and thereby provide absolute comparability of results. Method standardization is essential, as companies replace LAL/TAL with rFC and perform the required method validation according to USP <1225> Validation of Compendial Procedures, USP <85> Bacterial Endotoxins Test and other harmonized and/or equivalent global compendial chapters (8).
In addition to eliminating the animal source, rFC tests also provide fast and easy workflows and consistent results. There are rFC test systems available for a whole spectrum of applications, from high throughput testing of water to robust solutions for challenging sample matrices. Higher lot-to-lot consistency, the removal of the possibility of cross-reaction with ß-glucan and state-of-the-art sensitivity down to 0.001 EU/mL are additional test advantages. Another important gain with rFC is the lowered rate of invalid results; users report routine testing results without CV fails over extended periods, with positive product control recoveries constantly running close to 100%.
In the 2012 Guidance Q&A for endotoxin testing, the U.S. FDA included rFC as an alternative method (9). Since then, both the European and Japanese pharmacopoeias have taken additional actions. In 2016, the European Pharmacopoeia described rFC as an alternative method in Ph. Eur. Chapter 5.1.10 (10) and is now moving further ahead. In a press release dated April 5, 2018, the European Directorate for the Quality of Medicines announced that the European Pharmacopoeia Commission will “work on a general chapter on a test for bacterial endotoxins using recombinant Factor C, avoiding the use of a reagent coming from endangered species (horseshoe crab)” (11). Having a general chapter describing the method is particularly important in light of the increased number of companies using rFC and submitting drug master files. In addition, the Japanese Pharmaceuticals and Medical Devices Agency is conducting important collaborative studies on rFC as compared to LAL, which has resulted in an initial publication demonstrating equivalence (12). Ecological concerns and environmental sustainability are at the forefront of much research these days. The use of rFC as an alternative endotoxin testing method is one-way companies can address the concerns of an ever-growing environmentally conscious public.
[Editor’s Note: The author’s company, bioMérieux, will be exhibiting at the 13th Annual PDA Global Conference on Pharmaceutical Microbiology, the 2018 PDA Endotoxins Workshop and the 2018 PDA Europe Pharmaceutical Microbiology conference.]
1. Hoffman, W. D., and Natanson, C., “The Role of Endotoxin in Bacterial Septic Shock,” In Yearbook of Intensive Care and Emergency Medicine 1993. Yearbook of Intensive Care and Emergency Medicine 1993, Vincent JL. (eds), 49-63; Springer, Berlin, Heidelberg
2. Williams, K. L., “The Emerging View of Endotoxin as an IIRMI.” BioPharm International 28 (Feb. 1, 2016) 24–31.
3. Munro, M., “What’s killing the world’s shorebirds?” Nature 541 (2017): 16–20. 4. Smith, D.R., et al., “Limulus polyphemus, American Horseshoe Crab,” The IUCN Red List of Threatened Species.
5. Iwanaga, S., et al., “Haemolymph coagulation in Limulus,” In Microbiology. Leive L., et al. (eds), American Society of Microbiology, Washington, pp. 29–32 (1985).
6. Ding, J. L., Ho, B., “Assays for endotoxin,” United States Patent US 6,645,724 B1 (date of patent November 11, 2003)
7. Williams, K.L., "Specificity in the recombinant factor C test for endotoxin." European Pharmaceutical Review (April 26, 2018)
8. Bolden, J. S., and Smith, K. R., “Application of recombinant Factor C reagent for the detection of bacterial endotoxins in pharmaceutical products,” PDA Journal of Pharmaceutical Science and Technology 71 (2017):405-412.
9. Guidance for Industry: Pyrogen and Endotoxins Testing: Questions and Answers, U.S. FDA, June 2012
10. European Pharmacopoeia, “Chapter 5.1.10. Guidelines for using the test for bacterial endotoxins"
11. European Directorate for the Quality of Medicines, “Outcome of the 160th Session of the European Pharmacopoeia Commission,” Press Release.
Kikuchi, Y., et al., “Collaborative Study on the Bacterial Endotoxins Test Using Recombinant Factor C-based Procedure for Detection of Lipopolysaccharides,” Pharmaceutical and Medical Device Regulatory Science 48 (2017): 252-260.