USP <85> Bacterial Endotoxin

USP <85> Bacterial Endotoxin

Endotoxin contamination of a product results from the presence of Gram negative bacteria or components of their cell wall and can occur as a result of inadequate manufacturing controls.  Raw material components, containers, closures, manufacturing equipment and water systems are among the areas to address in establishing endotoxin control.  Adequate cleaning, drying, and storage of equipment as well as routine monitoring of water systems and incoming materials can help to ensure that the manufacturing process does not contribute endotoxins to the final product.

USP Chapter <85> Bacterial Endotoxins Test (BET) is an in-vitro assay to detect and quantity Gram negative bacteria, the source of endotoxin.  The BET is performed as part of lot release testing for injectable pharmaceutical products and medical devices with direct or indirect contact to the cardiovascular system, lymphatic system or cerebrospinal fluid.  The assay is also used as a screening test for incoming components and as a Quality Control (QC) test for water used in the manufacturing process.  

There are three methods used for this test: the semi-quantitative Gel-clot method, the quantitative Kinetic Turbidimetric method and the quantitative Kinetic Chromogenic method.

Study Outline

A liquid sample or sample extract prepared from a medical device or pharmaceutical product  is assayed using Limulus Amebocyte Lysate (LAL). LAL is a reagent made from the blood of the horseshoe crab and in the presence of bacterial endotoxins, forms a clot in the Gel Clot method a change in turbidity in the Turbidimetric method and color change in the Chromogenic method. The test sample response is compared to a standard curve made from known endotoxin concentrations. All tests are performed in duplicate. A positive product control and negative control are included as part of each assay.

Prior to conducting the BET assay each product must be validated to demonstrate that the product does not interfere with the ability to detect endotoxins. This is accomplished with the positive product control (also called the spike recovery) for the kinetic test methods (Turbidimetric and Chromogenic), and with a separate inhibition and enhancement assay for the gel-clot test.

Products requiring a treatment such as reconstitution in a solvent other than water, heat denaturing, centrifugation or filtration should be validated to demonstrate that the treatment does not result in the loss of endotoxins. This is accomplished by inoculating the sample with endotoxin, subjecting the inoculated sample to the chosen treatment then testing for endotoxin recovery. 

Endotoxin limits for pharmaceutical products are specified in individual USP monographs and are based on the maximum dose that can be administered in a 1-hour time period.  The limit for medical devices is not more than 20.0 EU/device for devices with blood contact and not more than 2.15 EU/device for devices with cerebrospinal fluid contact.

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