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.

Effects of Diet on the Biochemical Properties of Limulus Amebocyte Lysate From Horseshoe Crabs in an Aquaculture Setting

“…Divalent cations play an essential role in stabilizing LPS dimerization via cross-links, which is thought to be required for LPS biological activity. In the presence of cation chelating agents in vitro, LPS dimers are liberated into monomers, which have been shown to be unresponsive to LAL (Tsuchiya, 2019). In this study, aquaculture LAL was prepared following standard SOPs; whereby, amebocytes were washed in 0.5 M NaCl prior to lysis in water.…”

Improved endotoxin removal using ecofriendly detergents for intensified plasmid capture

“…20 However, low endotoxin recovery (LER) is an issue with this assay, which can result in a false negative. 21,22 Alkaline lysis treatment of fermented plasmid harvest releases endotoxins into the lysate. While release of endotoxins and other cellular impurities can be minimized by short incubation times at extreme pH and avoiding high mechanical mixing stress during cell lysis, downstream purification steps must be implemented for endotoxin clearance.…”

Low endotoxin recovery and its impact on endotoxin detection

“…Tsuchiya [27] Charles River The reduction of activity of LPS is due to the replacement of LPS molecules on the surface of aggregates by detergent molecules Orving et al [28] FUJIFILM Diosynth Biotechnologies, Biogen, Microcoat Biotechnologie GmbH, University of Southern Denmark Lauryldimethylamine oxide (LDAO), a component used in the virus inactivation step alone, could lead to a LER effect. Unlike the traditional LER effect that is time-dependent, the LDAO-induced LER is instant and time-independent Bu et al [29] BeiGene Guangzhou Biologics Manufacturing Co., Ltd.…”

“…[27][28][29] In 2019, Tsuchiya attempted to elucidate the mechanism of LER. [27] In this investigation, the relationship was studied between the change of particle sizes (measured by dynamic light scattering) and F I G U R E 4 Schematic illustration of the proposed physical state change of LPS during the LER event. A, LPS tends to form aggregates in aqueous solution due to its amphiphilic nature.…”

“…After the concept of LER was introduced, research groups have tried to address the LER phenomenon as shown in Table 1. [19][20][21][22][23][24][25][26][27][28][29] T A B L E 1 Selected studies of the low endotoxin recovery phenomenon…”