USP <85> Bacterial Endotoxins

Endotoxins are released by gram-negative bacteria; residual endotoxins may remain in areas where bacteria has been killed. The United States Pharmacopeia 47, General Chapter 85 Bacterial Endotoxins Test (BET) is designed to determine the concentration of bacterial endotoxins within a product. It is also known as the Limulus Amebocyte Lysate (LAL) test.

In 1980 the Federal Register recommended guidelines for determining endotoxin concentrations using the LAL test. These guidelines were revised and officially adopted by the FDA in 1983, and the test itself has been continuously harmonized with the European Pharmacopeia and the Japanese Pharmacopeia since 2008.

The test has three different techniques that can be used to ascertain a product’s endotoxin concentration with lysate. They are the Gel-Clot Technique, Turbidimetric Technique, and Chromogenic Technique. Microchem currently provides testing with the Chromogenic version of USP <85> via Pierce’s Chromogenic Endotoxin Quant Kit. The test is performed by generating a standard curve from prepared endotoxin standard solutions using their known concentrations and the optical density obtained by testing personnel. The equation from the standard curve is used to determine the endotoxin concentration of the product from its optical density.

Summary of the Test Procedure and Important Parameters

• At least three standard endotoxin solutions are prepared according to the manufacturer’s instructions.
• A predetermined volume of reconstituted lysate is added to each standard solution and test sample and allowed to remain undisturbed for a predetermined amount of time.
• At the end of the lysate’s time, the chromogenic substrate is added to each solution and allowed to remain undisturbed for a predetermined period of time. The opaqueness of the color is based on the endotoxin concentration.
  • Once that time is completed, acetic acid is used to stop the reaction.
• The optical density of each solution is determined at 405 nm.
• The standard endotoxin solutions are used to generate a standard curve. This standard curve is used to interpolate the endotoxin concentration of the test sample solutions from their optical densities.
  • The endotoxin concentration of the test sample solution must be below the endotoxin limit.
• To determine if the product contains any factors that would interfere with the measuring of endotoxins, the test outlined above is repeated with a test sample solution that has a specific amount of endotoxins introduced to it.

Sources:

United States Pharmacopeia. (2012, December 01). <85> Bacterial Endotoxins. https://www.usp.org/harmonization-standards/pdg/general-methods/bacterial-endotoxins

U.S. Food and Drug Administration. (2014, November 17). Bacterial Endotoxins/Pyrogens. https://www.fda.gov/inspections-compliance-enforcement-and-criminal-investigations/inspection-technical-guides/bacterial-endotoxinspyrogens