Development and validation of a targeted LC-MS/MS quantitation method to monitor cell culture expression of tetanus neurotoxin during vaccine production
| Autor: | Eric Abachin, Melissa Vanhamme, Alexandre Dobly, Sylvie Uhlrich, Celine Vanhee, Raphael Esson, Jean-François Dierick, Bruce Carpick, Antoine Francotte |
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| Rok vydání: | 2022 |
| Předmět: |
Microbiological culture
Clostridium tetani medicine.disease_cause 01 natural sciences Analytical Chemistry Microbiology 03 medical and health sciences Tetanus Toxin Tandem Mass Spectrometry medicine 030304 developmental biology Bacteriological Techniques 0303 health sciences Tetanus Toxin Chemistry 010401 analytical chemistry Toxoid Metalloendopeptidases Biomolecules (q-bio.BM) medicine.disease 0104 chemical sciences 3. Good health Vaccination body regions Quantitative Biology - Biomolecules Cell culture Tetanus vaccine FOS: Biological sciences Chromatography Liquid medicine.drug |
| DOI: | 10.48550/arxiv.2201.08447 |
| Popis: | The tetanus neurotoxin (TeNT) is one of the most toxic proteins known to man, which prior to the use of the vaccine against the TeNT producing bacteria Clostridium tetani, resulted in a 20 % mortality rate upon infection. The clinical detrimental effects of tetanus have decreased immensely since the introduction of global vaccination programs, which depend on sustainable vaccine production. One of the major critical points in the manufacturing of these vaccines is the stable and reproducible production of high levels of toxin by the bacterial seed strains. In order to minimize time loss, the amount of TeNT is often monitored during and at the end of the bacterial culturing. The different methods that are currently available to assess the amount of TeNT in the bacterial medium suffer from variability, lack of sensitivity, and/or require specific antibodies. In accordance with the consistency approach and the three Rs (3Rs), both aiming to reduce the use of animals for testing, in-process monitoring of TeNT production could benefit from animal and antibody-free analytical tools. In this paper, we describe the development and validation of a new and reliable antibody free targeted LC-MS/MS method that is able to identify and quantify the amount of TeNT present in the bacterial medium during the different production time points up to the harvesting of the TeNT just prior to further upstream purification and detoxification. The quantitation method, validated according to ICH guidelines and by the application of the total error approach, was utilized to assess the amount of TeNT present in the cell culture medium of two TeNT production batches during different steps in the vaccine production process prior to the generation of the toxoid. The amount of TeNT generated under different physical stress conditions applied during bacterial culture was also monitored. Comment: manuscript accepted for publication by talanta (DOI 10.1016/j.talanta.2021.122883). In total |
| Databáze: | OpenAIRE |
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