Microbiology

Endotoxin detection using the LAL test or rFC test verifies the safety, namely the absence of endotoxins, of sterile pharmaceutical products for human use. Furthermore, the detection of endotoxins in non-sterile products, as well as in the analysis of pharmaceutical water, plays an important role.

The detection of bacterial endotoxins originating from Gram-negative bacteria is still mainly performed using amebocyte lysate (LAL) obtained from horseshoe crab blood (Limulus polyphemus or Tachypleus tridentatus). The corresponding chapters have been harmonized between the European Pharmacopoeia ((EP) 2.6.14.), the United States Pharmacopoeia ((USP) <85>), and the Japanese Pharmacopoeia ((JP) 4.01). Endotoxins are toxic to humans and/or animals and cause a pyrogenic reaction (increase in body temperature). For this reason, it is important that drugs and medical devices that are either injected or implanted be tested for their pyrogen and thus endotoxin content.

The detection of endotoxins is also used for the evaluation of medical devices such as disposable devices and implants. For this purpose, the test product is extracted with pyrogen-free water (PFW) and tested for the presence of endotoxin in the extracts.

Several methods are available for performing the endotoxin test, including the in vivo pyrogen test (rabbit pyrogen testing, RPT) and the in vitro Limulus amebocyte lysate test, or LAL test, and the recombinant factor C test, or rFC test. Several methods are available to perform the LAL assay, such as the gel-clot method, the kinetic turbidimetric (KTA) method, and the kinetic chromogenic (KCA) method. According to the European Commission (Ph. Eur.), the detection of pyrogens other than endotoxins should no longer be performed using the rabbit pyrogen test, but should be completely replaced by the monocyte activation test (MAT) by 2026.

Eurofins BioPharma Product Testing DACH is also happy to support you if you have a need for MAT testing.

TEST PRINCIPLE of the LAL test:

LAL kinetic chromogenic (KCA) assay and kinetic turbidimetric (KTA) assay.

Eurofins BioPharma Product Testing DACH offers the performance of both kinetic chromogenic assays and kinetic turbidimetric assays. The chromogenic method involves an enzymatic reaction between endotoxin and the modified LAL lysate, resulting in the production of a yellow color in the presence of endotoxin. The intensity of color production is directly related to the amount of endotoxin present in the sample. Therefore, we can use endotoxin standards to calculate the value of endotoxin present in or on the product. Some products have an inherent color that would interfere with measurement by KCA assay, so the turbidimetric method can be used. In this case, KTA lysate is used and reaction with endotoxin causes the solution to become turbid, allowing quantification of endotoxin content independent of color. Both methods use objective measurements to determine endotoxin content and are quantitative in nature and therefore preferable to gel-clot. 

LAL Gel-Clot Assay

The gel-clot assay was the original LAL method. It is a qualitative or semi-quantitative test used to screen for the presence of endotoxins. The formation of a solid gel indicates the presence of endotoxins in the tested sample. 

TEST PRINCIPLE of the rFC test, an alternative to the LAL test.

The EDQM has published the European Pharmacopoeia (EP) version 10.3, which now includes the new chapter 2.6.32 "Test for bacterial endotoxins using recombinant factor C (rFC)". The factor C assay is therefore an animal-free and good alternative for the detection of endotoxins in Europe. The USP is also working on a draft chapter (USP <1085.1>) on the use of recombinant factor C.

The rFC enzyme, cloned from the horseshoe crab, is recombinant and when endotoxin binds to recombinant factor C, the activated rFC enzyme cleaves a synthetic fluorogenic substrate, causing the solution to fluoresce. We offer different rFC test kits and are independent of manufacturers.

Eurofins BioPharma Product Testing DACH is able to support you with well set up comparative studies to enable the possible change of method - from LAL to rFC for the detection of endotoxins in your products. 

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