Endotoxin detection by LAL and rFC test at Eurofins BioPharma Product Testing DACH
Endotoxin detection by LAL test or rFC test checks the safety, namely absence of endotoxins, sterile pharmaceutical products for human consumption. In addition, 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, which originate from gram-negative bacteria, is mainly carried out using amoevocyte lysate (LAL), which is obtained from horseshoe crab blood (Limulus polyphemus or Tachypleus tridentatus). The corresponding chapters have been harmonised between the European Pharmacopoeia ((EP) 2.6.14.), the Pharmacopoeia of the United States ((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 are 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. To do this, 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 carrying out the endotoxin test, including the in-vivo pyrogen test (Rabbit Pyrogen Testing, RPT) and the in-vitro Limulus amoevocyte lysate test, LAL test for short, and the recomibinate factor C test, rFC test for short. Various methods are available for performing the LAL test, 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 that are not endotoxins should no longer be carried out via the rabbit pyrogenic test, but will 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.
PRINCIPLE OF LAL TEST:
Kinetic Chromogenic method (KCA) and Kinetic Turbidimetric method (KTA)
Eurofins BioPharma Product Testing is able to perform both the kinetic chromogenic and turbidimetric assays. The chromogenic method involves an enzymatic reaction between the endotoxin and lysate which results in the production of a yellow colour in the presence of endotoxin. The intensity of the colour production is directly linked to the quantity of endotoxin present in the sample. With the kinetic variation of the assay, the time of onset of the colour reaction is measured. Therefore, with the use of endotoxin standards we are able to calculate the value of endotoxin present in or on the product. Some products are of a colour that would interfere with this form of testing, and so the turbidimetric method can be used to avoid any such interference. In this case a different lysate is used and the reaction with endotoxin results in the solution becoming turbid, thus allowing quantitation of endotoxin content without relying on the colour present. Both methods are equally effective in obtaining the endotoxin content in a product, but often, one is more suitable than the other. Both methods use objective measurements to determine endotoxin content and are quantitative in nature. These tests can be carried out relatively quickly, and results can be available within one week of sample receipt.
LAL Gel Clot Assay method
The gel clot assay was the original LAL method. It is a qualitative or semi-quantitative test that is used to screen for the presence of endotoxins. The formation of a firm gel indicates the presence of endotoxins in the tested sample.
SAMPLE REQUIREMENTS for the LAL Test:
Product validation, inhibition or enhancement properties of the materials on the test system.
SAMPLE REQUIREMENTS [FDA and AAMI Guidelines]:
Samples from three (3) production lots should be tested for inhibition and enhancement before this test is considered validated for use with the test product. The number of samples required for each lot is the same as *below.
NOTE: Validation testing can be performed at the same time and on the same samples as the lot release (finished product) testing.
Endotoxin testing of water system samples or other liquids.
SAMPLE REQUIREMENTS:
1 mL in a sealed endotoxin-free polystyrene or glass container.
Quantitative determination of endotoxin level for finished devices or other materials.
*SAMPLE REQUIREMENTS [FDA Guidelines]:
For lots of less than 30 units – 2 sample devices
For lots of 30-100 units – 3 sample devices
For lots of 101 units or greater – 3% of lot, up
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 contains 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 compendial alternative to 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 produced recombinate and when endotoxin binds to recombinant factor C, the activated rFC enzyme breaks down a synthetic fluorogenic substrate, causing a fluoresce signal. We offer different rFC test kits and are independent of the manufacturers.
Eurofins BioPharma Product Testing DACH is able to support you with well-prepared comparative studies to enable the possible change of method - from LAL to rFC for the detection of endotoxins in your products.
