Detection of Lipopolysaccharides in Phospholipids and Liposomes Using the Limulus Test

Abstract

LPS (lipopolysaccharides) represent a feared pyrogenic impurity in parenterals and raw materials used for their production. In liposome dispersions detection of LPS via the standard Limulus amoebocyte lysate (LAL) test was proved unreliable in presence of phospholipids (liposomes). Attempts were made either to eliminate or inactivate disturbances of the LAL test by phospholipid(s). Common methods to overcome inhibition of the test, such as dilution of the sample, removal of the inhibiting substance by centrifugation or its inactivation by addition of detergents, were found not successful when LPS was present in liposome membrane-bound form. Another means to remove inhibiting substances is ultrafiltration. Ultrafiltration of aqueous lipid dispersions cannot be performed due to clogging of the filter membrane. Ultrafiltration upon addition of organic solvents turned out to be difficult due to the very limited resistance of celluloseacetate filter membranes against these solvents. Nevertheless a test protocol for detection of lipopolysaccharides in phospholipids and liposomes could be worked out and validated. It comprises dissolution of the phospholipid or liposomesNin pyrogen-free ethanol/water mixtures, ultrafiltration through pyrogen-free Ultrasart D20 units (Sartorius AG, Gottingen), reconstitution of the residue in pyrogen-free water or buffer and LAL testing. This procedure has been shown to allow for quantitative detection of LPS from minute amounts of 0.5 EU/ml up to very high amounts of 1000 EU/ml in liposomes in a reliable and reproducible manner. Recovery of LPS in all cases was fairly high i.e. one to two dilution steps below the theoretical LPS content.