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Bacterial Endotoxin Testing—Fast Endotoxin Masking Kinetics in the Presence of Lauryldimethylamine Oxide

For release of parenteral drug products, bacterial endotoxin testing is one of a panel of necessary tests. In order to ensure the validity of such tests, various controls are performed, including demonstration of compendial method suitability or method qualification. In addition to compendial suitab...

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Autores principales: Bech Ørving, René, Carpenter, Bill, Roth, Steffen, Reich, Johannes, Kallipolitis, Birgitte H., Sonne-Hansen, Jacob
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694283/
https://www.ncbi.nlm.nih.gov/pubmed/33158205
http://dx.doi.org/10.3390/microorganisms8111728
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author Bech Ørving, René
Carpenter, Bill
Roth, Steffen
Reich, Johannes
Kallipolitis, Birgitte H.
Sonne-Hansen, Jacob
author_facet Bech Ørving, René
Carpenter, Bill
Roth, Steffen
Reich, Johannes
Kallipolitis, Birgitte H.
Sonne-Hansen, Jacob
author_sort Bech Ørving, René
collection PubMed
description For release of parenteral drug products, bacterial endotoxin testing is one of a panel of necessary tests. In order to ensure the validity of such tests, various controls are performed, including demonstration of compendial method suitability or method qualification. In addition to compendial suitability testing, quality control (QC) sample hold-time studies are requested by authorities like the Food and Drug Administration (FDA) as described in “Guidance for Industry: Pyrogen and Endotoxins Testing.” It is requested to be determine whether the ability to detect endotoxins can be affected by storage and handling of the sample to be tested. To accomplish these studies, endotoxin is introduced or spiked into the undiluted product and held for a certain period of time in process-representative containers. This time period reflects procedural maximum QC sample hold time from sampling until analysis. Inadequate detection of endotoxin can be caused by adsorption of endotoxin to container surfaces or molecular masking effects, in which the binding sites on the endotoxin molecules are prevented from triggering the enzymatic cascade necessary in the assay, are obscured. The endotoxin may form macromolecular structures, such as sheets or blebs, or the binding sites may otherwise be rendered unavailable due to the sample matrix composition. In either case, the endotoxin assay may yield falsely low results if and when masking occurs. In this work, the QC sample hold times of different in-process controls within the production process of a biopharmaceutical product were analyzed. One out of eight different samples showed a strong masking of endotoxin. Analysis of the sample composition revealed that either kifunensine, mycophenolic acid (MPA), or lauryl-N, N-dimethylamine oxide (LDAO) was responsible for masking. Further analysis clearly identified LDAO as the root cause for masking. A novel one-step mechanism for LDAO-induced endotoxin masking is proposed. The principle is similar to an already-proposed two-step mechanism for endotoxin masking, but the LDAO case combines these two steps: the disturbance of the salt bridges and hydrophobic interactions with LPS in one molecule. These molecular interactions occur quickly when both endotoxin and LDAO are present in the same matrix. Thus, depending on the masking agents, low endotoxin recovery (LER) can occur regardless of the QC sample hold duration.
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spelling pubmed-76942832020-11-28 Bacterial Endotoxin Testing—Fast Endotoxin Masking Kinetics in the Presence of Lauryldimethylamine Oxide Bech Ørving, René Carpenter, Bill Roth, Steffen Reich, Johannes Kallipolitis, Birgitte H. Sonne-Hansen, Jacob Microorganisms Article For release of parenteral drug products, bacterial endotoxin testing is one of a panel of necessary tests. In order to ensure the validity of such tests, various controls are performed, including demonstration of compendial method suitability or method qualification. In addition to compendial suitability testing, quality control (QC) sample hold-time studies are requested by authorities like the Food and Drug Administration (FDA) as described in “Guidance for Industry: Pyrogen and Endotoxins Testing.” It is requested to be determine whether the ability to detect endotoxins can be affected by storage and handling of the sample to be tested. To accomplish these studies, endotoxin is introduced or spiked into the undiluted product and held for a certain period of time in process-representative containers. This time period reflects procedural maximum QC sample hold time from sampling until analysis. Inadequate detection of endotoxin can be caused by adsorption of endotoxin to container surfaces or molecular masking effects, in which the binding sites on the endotoxin molecules are prevented from triggering the enzymatic cascade necessary in the assay, are obscured. The endotoxin may form macromolecular structures, such as sheets or blebs, or the binding sites may otherwise be rendered unavailable due to the sample matrix composition. In either case, the endotoxin assay may yield falsely low results if and when masking occurs. In this work, the QC sample hold times of different in-process controls within the production process of a biopharmaceutical product were analyzed. One out of eight different samples showed a strong masking of endotoxin. Analysis of the sample composition revealed that either kifunensine, mycophenolic acid (MPA), or lauryl-N, N-dimethylamine oxide (LDAO) was responsible for masking. Further analysis clearly identified LDAO as the root cause for masking. A novel one-step mechanism for LDAO-induced endotoxin masking is proposed. The principle is similar to an already-proposed two-step mechanism for endotoxin masking, but the LDAO case combines these two steps: the disturbance of the salt bridges and hydrophobic interactions with LPS in one molecule. These molecular interactions occur quickly when both endotoxin and LDAO are present in the same matrix. Thus, depending on the masking agents, low endotoxin recovery (LER) can occur regardless of the QC sample hold duration. MDPI 2020-11-04 /pmc/articles/PMC7694283/ /pubmed/33158205 http://dx.doi.org/10.3390/microorganisms8111728 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Bech Ørving, René
Carpenter, Bill
Roth, Steffen
Reich, Johannes
Kallipolitis, Birgitte H.
Sonne-Hansen, Jacob
Bacterial Endotoxin Testing—Fast Endotoxin Masking Kinetics in the Presence of Lauryldimethylamine Oxide
title Bacterial Endotoxin Testing—Fast Endotoxin Masking Kinetics in the Presence of Lauryldimethylamine Oxide
title_full Bacterial Endotoxin Testing—Fast Endotoxin Masking Kinetics in the Presence of Lauryldimethylamine Oxide
title_fullStr Bacterial Endotoxin Testing—Fast Endotoxin Masking Kinetics in the Presence of Lauryldimethylamine Oxide
title_full_unstemmed Bacterial Endotoxin Testing—Fast Endotoxin Masking Kinetics in the Presence of Lauryldimethylamine Oxide
title_short Bacterial Endotoxin Testing—Fast Endotoxin Masking Kinetics in the Presence of Lauryldimethylamine Oxide
title_sort bacterial endotoxin testing—fast endotoxin masking kinetics in the presence of lauryldimethylamine oxide
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694283/
https://www.ncbi.nlm.nih.gov/pubmed/33158205
http://dx.doi.org/10.3390/microorganisms8111728
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