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Int. J. Mol. Sci. 2017, 18(12), 2737; https://doi.org/10.3390/ijms18122737

Biophysical Analysis of Lipopolysaccharide Formulations for an Understanding of the Low Endotoxin Recovery (LER) Phenomenon

1
Forschungszentrum Borstel, Leibniz-Zentrum für Medizin und Biowissenschaften, Parkallee 1-40, D-23845 Borstel, Germany
2
Brandenburg Antiinfektiva GmbH, Parkallee 10b, D-23845 Borstel, Germany
3
Pharmaceutical Development & Supplies, F. Hoffmann-La Roche Ltd., 4070 Basel, Switzerland
4
Roche Diagnostics GmbH, Nonnenwald 2, Roche Diagnostics GmbH, 82377 Penzberg, Germany
*
Author to whom correspondence should be addressed.
Received: 29 November 2017 / Revised: 11 December 2017 / Accepted: 13 December 2017 / Published: 16 December 2017
(This article belongs to the Special Issue Lipopolysaccharides (LPSs))
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Abstract

Lipopolysaccharides (LPS, endotoxin) are complex and indispensable components of the outer membrane of most Gram-negative bacteria. They represent stimuli for many biological effects with pathophysiological character. Recombinant therapeutic proteins that are manufactured using biotechnological processes are prone to LPS contaminations due to their ubiquitous occurrence. The maximum endotoxin load of recombinant therapeutic proteins must be below the pyrogenic threshold. Certain matrices that are commonly used for recombinant therapeutic proteins show a phenomenon called “Low Endotoxin Recovery (LER)”. LER is defined as the loss of detectable endotoxin activity over time using compendial Limulus amebocyte lysate (LAL) assays when undiluted products are spiked with known amount of endotoxin standards. Because LER poses potential risks that endotoxin contaminations in products may be underestimated or undetected by the LAL assay, the United States (U.S.) Food and Drug Administration’s (FDA’s) Center for Drug Evaluation and Research (CDER) has recently started requesting that companies conduct endotoxin spike/hold recovery studies to determine whether a given biological product causes LER. Here, we have performed an analysis of different LPS preparations with relevant detergents studying their acyl chain phase transition, their aggregate structures, their size distributions, and binding affinity with a particular anti-endotoxin peptide, and correlating it with the respective data in the macrophage activation test. In this way, we have worked out biophysical parameters that are important for an understanding of LER. View Full-Text
Keywords: endotoxin; lipopolysaccharide; Low Endotoxin Recovery; phase transitions; polysorbate; LPS aggregates; Small Angle X-ray Scattering; MAT; LAL and LER endotoxin; lipopolysaccharide; Low Endotoxin Recovery; phase transitions; polysorbate; LPS aggregates; Small Angle X-ray Scattering; MAT; LAL and LER
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Correa, W.; Brandenburg, K.; Zähringer, U.; Ravuri, K.; Khan, T.; von Wintzingerode, F. Biophysical Analysis of Lipopolysaccharide Formulations for an Understanding of the Low Endotoxin Recovery (LER) Phenomenon. Int. J. Mol. Sci. 2017, 18, 2737.

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