Bacterial endotoxin assays: LAL and rFC

endotoxin testing

Endotoxin testing is an important standard of many proteins’ quality control reporting. Verifying the amount of endotoxin in a sample of protein is critical to ensure that it is safe for research and therapeutic applications.

Why perform endotoxin testing?

Endotoxins are a type of pyrogen, meaning that they cause a fever response with enough volume in an organism. These lipopolysaccharides are found in the cell walls of gram-negative bacteria. It is crucial to know if the level of endotoxin present in a sample is below a safe threshold, especially for injectable pharmaceuticals and medical implants. Even for research purposes, it is important to avoid toxic materials that could confound results, especially in cell culture.

Measuring endotoxin levels

Endotoxin activity levels are reported in EU/mL (endotoxin units). One EU is roughly equivalent to 0.1-0.2 ng endotoxin/mL of solution. The FDA sets restrictions on acceptable levels in devices and non-oral drugs, but there is not a set standard for research purposes. Recombinant proteins for research tend to have <1.0 EU/ug. TriAltus’ human growth hormone has <0.1 EU/ug and Cas9 has <0.3 EU/ug as measured by the rFC method. 

Bacterial endotoxin testing methods

Endotoxin testing began with the rabbit pyrogen method in which a rabbit would be injected with the sample in question. If the rabbit showed a fever response, the sample was assumed to contain an unacceptable level of endotoxin. However, this method was too non-specific, and the LAL test quickly took hold as the standard procedure.

LAL (Limulus amoebocyte lysate) testing originates from the blood of horseshoe crabs. The presence of endotoxin in the lysate triggers proteins such as recombinant factors C, G, and B in a signaling cascade that activates a clotting enzyme.  It is the cascade that allows for the extreme sensitivity of the test. To test for endotoxin, samples are mixed with LAL, and coagulation is measured either through a chromogenic or a turbidimetric assay. These tests can register up to 0.01 EU/mL.

Despite being the most widely used means of endotoxin testing, LAL has several downsides. Horseshoe crabs and their blood are highly limited resources bordering on endangered. LAL has natural variations between samples due to differences among the population of horseshoe crabs. Additionally, the clotting cascade can be initiated by off-target effects and decrease the accuracy of the assay. These factors make LAL ripe for improvement.

lal rfc endotoxin testing

Source: Lonza Pyrogene rFC testing

Recombinant Factor C (rFC) testing

A more sustainable and accurate option for endotoxin testing is available in the form of recombinant factor C (rFC) testing. Recombinant factor C is one of the proteins naturally found in LAL that is the first enzyme in the clotting cascade. rFC uses only the factor C protein and a fluorescent marker to measure the level of endotoxin present in the sample. This approach simplifies the original LAL test and preserves horseshoe crab populations. rFC testing is gaining widespread acceptance and will be designated a compendial test to LAL by the European Pharmacopeia by January 2021.