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Endotoxin Detection of Pharmaceutical and Medical Devices


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Endotoxin Detection of Pharmaceutical and Medical Devices

Endotoxins, also known as pyrogens, are complex lipopolysaccharides which form an inherent fraction of the outer cell wall of all gram negative bacteria. Endotoxins are ubiquitous microbiological contaminants in biological and clinical products. Since they induce pyrogenic response and even terrible clinical response, endotoxins are considered as a key safety and quality issue for the pharmaceutical and medical devices at a long time. Therefore, endotoxin testing is required to ensure the biosafety of the end products.

The standard method for endotoxin detection is LAL test, which is based on amoebocyte lysate from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). In 1950s, it was discovered that the blood of the Atlantic horseshoe crab (Limulus polyphemus) forms clots in the presence of bacterial endotoxin. The reaction is caused by a clotting factor contained in amoebocytes. Based on the reaction, sensitive and accurate tests for endotoxin have successfully developed. There are three techniques for LAL test: the gel-clot technique, the turbidimetric technique and the chromogenic technique. The gel-clot assay can detect down to 0.03 EU/mL, while the turbidimetric assay and chromogenic assay can detect down to 0.01 EU/mL. In the event of doubt or dispute, the final decision is made upon the gel clot limit test.

1. Gel-Clot Technique

The gel-clot technique is based on the formation of clotting of the lysate in the presence of bacterial endotoxins in the test material. If a clot appears after the testing sample mixed with testing reagents, the result is positive. The minimum concentration of endotoxin required to form a clot under standard condition is the labeled sensitivity of the LAL reagents. The gel-clot assay is an endpoint method and can be used as a semi-quantitative method by testing serial dilutions.

Endotoxin Detection of Pharmaceutical and Medical Devices Fig 1. Gel-Clot assay

2. Turbidimetric technique

The turbidimetric technique is a photometric assay measuring increases in reactant turbidity induced by gel clot. There are two forms of turbidimetric assays, endpoint-turbidimetric assay and kinetic-turbidimetric assay. The endpoint turbidimetric assay is to determine the turbidity of the reaction mixture at the end of an incubation period, while the kinetic turbidimetric assay is to measure the time needed to reach a predetermined absorbance or transmission of the reaction mixture, or the rate of turbidity development.

3. Chromogenic Technique

The chromogenic technique employs a synthetic substrate that produce a color change at the presence of endotoxins. This technique can be classified as either an endpoint-chromogenic assay and a kinetic-chromogenic assay on the basis of the particular assay principle employed. The endpoint assay is to quantify the release of chromophore at the end of an incubation period, while the kinetic assay is to measure the time needed to reach a predetermined absorbance or transmission of the reaction mixture, or the rate of color development.

Endotoxin Detection of Pharmaceutical and Medical Devices Fig 2. Standard curve of endpoint chromogenic assay

Creative Biogene, as a global provider of biosafety testing, offers comprehensive endotoxin detection and removal services, and related products for pharmaceuticals, biopharmaceuticals and medical devices. All tests are performed by skillful staff under a strictly controlled environment. We are willing to serve customer with reliable products and services.

Reference:

  1. The United States Pharmacopeial Convention, <85>Bacterial Endotoxins Test, 2012,

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