Bacterial endotoxin testing plays a vital role in guaranteeing the safety of pharmaceuticals, medical devices, and other products that interact with the human body in the pharmaceutical and medical device industries. Chromak Research has extensive experience in performing this testing, with our experts adhering to the strict guidelines outlined in USP and USP. In this article, we are going to understand endotoxin’s description, its testing, importance of testing and its methods for testing.

What is Endotoxin and Bacterial Endotoxin Testing?

Endotoxins, which are lipopolysaccharides (LPS) located in the outer membrane of Gram-negative bacteria, have the potential to induce fever, inflammation, and various negative responses in humans. The assessment process revolves around identifying endotoxins through the utilization of Limulus Amebocyte Lysate (LAL), a substance obtained from the blood cells of the horseshoe crab (Limulus polyphemus). The LAL test is capable of detecting minute quantities of endotoxins and is highly regarded in the pharmaceutical sector for its precision and dependability.

The Bacterial Endotoxins Test (BET), also known as the LAL test, is used to detect and measure bacterial endotoxins. These endotoxins are found in the cell wall of gram-negative bacteria. The BET is essential for testing medical devices that come into contact with the cardiovascular, lymphatic, or cerebrospinal fluid systems, as well as injectable pharmaceutical products. Regular monitoring of water systems and materials is crucial to prevent endotoxins from contaminating the final product. The BET is sometimes called the pyrogen test because endotoxins can cause fever in mammals. However, it should not be confused with the rabbit pyrogen test described in USP chapter <151>. USP <85> mandates bacterial endotoxin testing for sterile injectables to ensure safety.

Importance of Bacterial Endotoxin Testing

Endotoxins in medical devices can be dangerous if undetected. They can cause adverse reactions like hemorrhagic shock, diarrhea, meningitis, and more. The Bacterial Endotoxin Test is used to measure endotoxins in gram-negative bacteria. It is necessary for devices that touch the cardiovascular or cerebrospinal systems. Testing for endotoxins is crucial to ensure patient safety, as they can lead to severe health issues and even death. Any device that directly or indirectly comes into contact with intravascular, intralymphatic, intrathecal, and/or intraocular systems must undergo Bacterial Endotoxin testing to ensure that the device’s endotoxin content is within the specified limits. Testing samples for bacterial endotoxins is crucial to ensure patient safety. Given the potential harmful effects of endotoxins on human and animal health, it is important to test medical devices, injectables, raw chemicals, API, excipients, process water, container closures, and other components that come into contact with the bloodstream or spinal fluid.

Testing methods of BET

Chromak Research provides dependable and precise Bacterial Endotoxins Testing (BET) <USP 85> techniques using either LAL or recombinant cascade reagent (rCR) to identify bacterial endotoxins in pharmaceutical industry and medical device manufacturing systems’ final products. Our offerings consist of gel-clot, kinetic turbidimetric, kinetic chromogenic, and recombinant cascade reagent (rCR) methods.

  • Kinetic Chromogenic:The kinetic chromogenic test involves mixing a sample with lysate. If endotoxins are present, the lysate will react with the endotoxin, causing a release of chromophore from a chromogenic peptide. A microplate reader is used to record the time it takes for the reaction mixture to reach a predetermined absorbance. The endotoxin content in the sample is then calculated using data from a predefined endotoxin standard.
  • Gel Clot: The test sample and lysate are combined in equal proportions within a tube and left to incubate. Following the incubation period, the tube is then inverted. Should endotoxin be detected, the mixture will coagulate, resulting in the gel settling at the base of the tube.
  • Kinetic Turbidimetric: By employing the kinetic turbidimetric test, the sample is combined with lysate. If endotoxins are present, the turbidity of the sample mixture will rise. The time taken to achieve a specific absorbance level in the reaction mixture is measured using a microplate reader. The endotoxin concentration in the sample is determined based on the relevant information obtained from a predetermined endotoxin standard.
  • Recombinant cascade reagent: This approach utilizes a recombinant form of the horseshoe crab factor C, the enzyme that responsible for coagulation in the LAL test. It presents a substitute for the LAL test that does not depend on the finite and ecologically delicate horseshoe crab blood.

Chromak Research boasts cutting-edge laboratory facilities that are fully equipped with various techniques and adhere to ISO 9001:2015 and ISO/IEC 17025:2017 standards. Specializing in Bacterial Endotoxin Testing for the pharmaceutical industry, we cater to manufacturers of sterile and non-sterile pharmaceutical products. Our laboratory conducts all necessary microbiological testing in compliance with USP, FDA/BAM guidelines. Our team of experts possesses the expertise and experience required for developing new methods and validating these type of microbiological testing procedures.