Characterization of a cell line, qualification, and comparison of automata to a manual method for cell counting / Jade Nichols

Public ISBD Titre : Characterization of a cell line, qualification, and comparison of automata to a manual method for cell counting Type de document : TFE / Mémoire Auteurs : Jade Nichols, Auteur ; Caroline Charlier, Directeur de la recherche Editeur : Montignies-sur-Sambre : Helha. Paramédical - Biologie médicale Année de publication : 2021 Note générale : Le fichier numérique de ce document est disponible uniquement pour les membres de la Haute Ecole Louvain-en-Hainaut ainsi que ses étudiants. Veuillez-vous connecter pour accéder à votre compte lecteur Langues : Anglais (eng) Index. décimale : TFE Bio Med TFE Biologie médicale Résumé : Cell culture is a rapidly expanding field. This is mainly due to the development of new technologies, such as the production of biomedicines. Exothera is a biotechnology firm which optimize process and manufacture’s viral vectors. The analytical team of their laboratory is responsible of supporting and confirming the production and purification parts of the production line. Hence, they need cells to determine whether the products meet the customer's expectations. To be able to use known cell lines in a production chain, it is necessary to know whether the cell banks that have been created from them are fit for purpose and behave identically to the original cell line. Therefore, determination of the cell doubling time and assessment of culture sterility are essential. Furthermore, as the bio-production range increases, there is a need to find ways to reduce the time spent on each step of the cell culture process. One such time-consuming step is manual cell counting, which is necessary as part of a cell passage to keep the cell culture alive. Manual counting could be replaced by an automated method. At Exothera, two automata are available: the iPrasens Norma XS and the Vi-CELL XR. To ensure that these two machines meet the Exothera’s requirements, it is necessary to qualify them by testing their linearity range and accuracy. This is the purpose of this thesis, a cell characterization, and a method qualification on two machines. The results obtained will make it possible to determine whether the cells can be used in an analytical laboratory, and whether the two automata can replace a manual method. Note de contenu : able of Contents Presentation of the company ...................................................................................................6 General introduction.................................................................................................................8 1 General context ..................................................................................................................9 1.1 Cell culture ..................................................................................................................9 1.2 Cell line characterization...........................................................................................13 1.2.1 The cell doubling time........................................................................................13 1.2.2 Biological contamination ...................................................................................14 1.3 Cell counting..............................................................................................................21 1.3.1 Bürker cell counting chamber............................................................................21 1.3.2 iPrasens Norma XS .............................................................................................24 1.3.3 The Vi-CELL XR....................................................................................................27 1.4 Qualification ..............................................................................................................30 1.4.1 Linearity .............................................................................................................30 1.4.2 Accuracy.............................................................................................................31 2 Objectives .........................................................................................................................33 3 Material and method........................................................................................................34 3.1 Cell characterization..................................................................................................34 3.1.1 Cell doubling time. .............................................................................................34 3.1.2 Sterility test ........................................................................................................39 3.1.3 Mycoplasma detection ......................................................................................43 3.2 Cell counting..............................................................................................................46 3.2.1 Determining the linearity, the range, the LOD and the LOQ.............................46 3.2.2 Determining the accuracy of the method .........................................................48 4 Results and discussion ......................................................................................................50 4.1 Cell characterization..................................................................................................50 5 4.1.1 The cell doubling time........................................................................................51 4.1.2 The sterility ........................................................................................................55 4.1.3 The mycoplasma ................................................................................................56 4.2 Automata’s qualification ...........................................................................................57 4.2.1 The linearity .......................................................................................................57 4.2.2 Accuracy based on qualification testing ............................................................64 4.2.3 Accuracy based on routine testing ....................................................................67 5 Conclusion and perspective..............................................................................................70 Abbreviations, glossary, and list of figures ............................................................................72 Bibliography.............................................................................................................................76 Annex .......................................................................................................................................79 Permalink : ./index.php?lvl=notice_display&id=100372 Characterization of a cell line, qualification, and comparison of automata to a manual method for cell counting [TFE / Mémoire] / Jade Nichols, Auteur ; Caroline Charlier, Directeur de la recherche . - Montignies-sur-Sambre : Helha. Paramédical - Biologie médicale, 2021. Le fichier numérique de ce document est disponible uniquement pour les membres de la Haute Ecole Louvain-en-Hainaut ainsi que ses étudiants. Veuillez-vous connecter pour accéder à votre compte lecteur Langues : Anglais (eng) Index. décimale : TFE Bio Med TFE Biologie médicale Résumé : Cell culture is a rapidly expanding field. This is mainly due to the development of new technologies, such as the production of biomedicines. Exothera is a biotechnology firm which optimize process and manufacture’s viral vectors. The analytical team of their laboratory is responsible of supporting and confirming the production and purification parts of the production line. Hence, they need cells to determine whether the products meet the customer's expectations. To be able to use known cell lines in a production chain, it is necessary to know whether the cell banks that have been created from them are fit for purpose and behave identically to the original cell line. Therefore, determination of the cell doubling time and assessment of culture sterility are essential. Furthermore, as the bio-production range increases, there is a need to find ways to reduce the time spent on each step of the cell culture process. One such time-consuming step is manual cell counting, which is necessary as part of a cell passage to keep the cell culture alive. Manual counting could be replaced by an automated method. At Exothera, two automata are available: the iPrasens Norma XS and the Vi-CELL XR. To ensure that these two machines meet the Exothera’s requirements, it is necessary to qualify them by testing their linearity range and accuracy. This is the purpose of this thesis, a cell characterization, and a method qualification on two machines. The results obtained will make it possible to determine whether the cells can be used in an analytical laboratory, and whether the two automata can replace a manual method. Note de contenu : able of Contents Presentation of the company ...................................................................................................6 General introduction.................................................................................................................8 1 General context ..................................................................................................................9 1.1 Cell culture ..................................................................................................................9 1.2 Cell line characterization...........................................................................................13 1.2.1 The cell doubling time........................................................................................13 1.2.2 Biological contamination ...................................................................................14 1.3 Cell counting..............................................................................................................21 1.3.1 Bürker cell counting chamber............................................................................21 1.3.2 iPrasens Norma XS .............................................................................................24 1.3.3 The Vi-CELL XR....................................................................................................27 1.4 Qualification ..............................................................................................................30 1.4.1 Linearity .............................................................................................................30 1.4.2 Accuracy.............................................................................................................31 2 Objectives .........................................................................................................................33 3 Material and method........................................................................................................34 3.1 Cell characterization..................................................................................................34 3.1.1 Cell doubling time. .............................................................................................34 3.1.2 Sterility test ........................................................................................................39 3.1.3 Mycoplasma detection ......................................................................................43 3.2 Cell counting..............................................................................................................46 3.2.1 Determining the linearity, the range, the LOD and the LOQ.............................46 3.2.2 Determining the accuracy of the method .........................................................48 4 Results and discussion ......................................................................................................50 4.1 Cell characterization..................................................................................................50 5 4.1.1 The cell doubling time........................................................................................51 4.1.2 The sterility ........................................................................................................55 4.1.3 The mycoplasma ................................................................................................56 4.2 Automata’s qualification ...........................................................................................57 4.2.1 The linearity .......................................................................................................57 4.2.2 Accuracy based on qualification testing ............................................................64 4.2.3 Accuracy based on routine testing ....................................................................67 5 Conclusion and perspective..............................................................................................70 Abbreviations, glossary, and list of figures ............................................................................72 Bibliography.............................................................................................................................76 Annex .......................................................................................................................................79 Permalink : ./index.php?lvl=notice_display&id=100372

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