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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| Format: | Article |
| Language: | English |
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MDPI AG
2020-11-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/8/11/1728 |
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| author | René Bech Ørving Bill Carpenter Steffen Roth Johannes Reich Birgitte H. Kallipolitis Jacob Sonne-Hansen |
| author_facet | René Bech Ørving Bill Carpenter Steffen Roth Johannes Reich Birgitte H. Kallipolitis Jacob Sonne-Hansen |
| author_sort | René Bech Ørving |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-10T15:05:50Z |
| format | Article |
| id | doaj.art-28dab64ba05d41468ca1a83de0953995 |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-10T15:05:50Z |
| publishDate | 2020-11-01 |
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| series | Microorganisms |
| spelling | doaj.art-28dab64ba05d41468ca1a83de09539952023-11-20T19:46:54ZengMDPI AGMicroorganisms2076-26072020-11-01811172810.3390/microorganisms8111728Bacterial Endotoxin Testing—Fast Endotoxin Masking Kinetics in the Presence of Lauryldimethylamine OxideRené Bech Ørving0Bill Carpenter1Steffen Roth2Johannes Reich3Birgitte H. Kallipolitis4Jacob Sonne-Hansen5FUJIFILM Diosynth Biotechnologies, 3400 Hillerød, DenmarkBiogen, Cambridge, MA 02142, USAMicrocoat Biotechnologie GmbH, 82347 Bernried, GermanyMicrocoat Biotechnologie GmbH, 82347 Bernried, GermanyDepartment of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, DenmarkFUJIFILM Diosynth Biotechnologies, 3400 Hillerød, DenmarkFor 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.https://www.mdpi.com/2076-2607/8/11/1728low endotoxin recoveryLERmaskinglimulus amoebocyte lysateLALendotoxin |
| spellingShingle | René Bech Ørving Bill Carpenter Steffen Roth Johannes Reich Birgitte H. Kallipolitis Jacob Sonne-Hansen Bacterial Endotoxin Testing—Fast Endotoxin Masking Kinetics in the Presence of Lauryldimethylamine Oxide Microorganisms low endotoxin recovery LER masking limulus amoebocyte lysate LAL endotoxin |
| 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 | low endotoxin recovery LER masking limulus amoebocyte lysate LAL endotoxin |
| url | https://www.mdpi.com/2076-2607/8/11/1728 |
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