Mechanism of Low Endotoxin Recovery Caused by a Solution Containing a Chelating Agent and a Detergent

Abstract

Mechanism of Low Endotoxin Recovery (LER) was investigated by observing the change in particle sizes and activity of endotoxin in LER solutions containing sodium citrate and polysorbate 20. Interestingly, endotoxin aggregates were not dispersed to be smaller particles under LER conditions according to the decease of the activity. This observation was different from previous predictions of the mechanism. Endotoxin aggregates were observed by Dynamic Light Scattering, and activity was measured by the Limulus amebocyte lysate test. Particles with sizes similar to endotoxin aggregates were always observed despite different remaining activity of spiked endotoxin. This observation differed from previously proposed mechanisms for LER that dispersed endotoxin aggregates were smaller in sizes. Based on the findings, a new mechanism of LER is proposed. Purified endotoxin forms non-lamellar cubic structures in water, and the endotoxin aggregates are reinforced by divalent cations. When purified endotoxin is added to LER solutions, divalent cations are removed from outer layer of endotoxin by the chelating agent, and the loss of divalent cations weakens the outside layer of the endotoxin aggregates. The endotoxin aggregation structure is maintained at low temperature, but endotoxin molecules on the surface of the endotoxin aggregates are gradually replaced with detergent molecules at higher temperature. This replacement reduces the surface area of the endotoxin, and cause reduction of activity of the endotoxin aggregates. The apparent sizes of the aggregates are not changed after the replacement.