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Proteomics Based Process and Cell Line Development Applied to A Proteomics Based Process and Cell Line Development Applied to a Mammalian Therapeutic Enzyme A thesis submitted to University College London for the degree of Doctor of Biochemical Engineering by Damiano Migani 2017 Department of Biochemical Engineering, University College London Bernard Katz Building, Gower Street London WC1E 6BT Declaration I, Damiano Migani, confirm that the work presented in this thesis is my own. Where information had been derived from other sources, I confirm that this has been indicated in the thesis. 2 Table of Contents Table of Contents ....................................................................................................................... 3 Table of Figures ......................................................................................................................... 9 Table of Tables ........................................................................................................................ 11 1 Abstract ............................................................................................................................. 14 2 Introduction ...................................................................................................................... 15 2.1 Impact statement ....................................................................................................... 15 2.2 Literature review ....................................................................................................... 16 2.2.1 Biopharmaceutical industry ............................................................................... 16 2.2.2 Biopharmaceutical global market ...................................................................... 17 2.2.3 Rare diseases: lysosomal glycogen storage disease type II. .............................. 18 2.2.4 The Product ........................................................................................................ 23 2.2.5 Production of material........................................................................................ 30 2.2.6 Proteases ............................................................................................................ 41 2.2.7 Detection of product and impurities .................................................................. 51 2.3 Objectives .................................................................................................................. 60 2.3.1 Production of stable CHO cell line for material generation. ............................. 60 2.3.2 Development of assays for detection of product in the bioprocess stream ........ 60 2.3.3 Bioprocess mimic............................................................................................... 60 2.3.4 Characterization of cell stress and its impact on protease production ............... 60 3 2.4 Project estimated timeline / Gantt chart .................................................................... 62 3 Materials and methods ...................................................................................................... 63 3.1 Cell line generation ................................................................................................... 63 3.1.1 Cloning ............................................................................................................... 63 3.1.2 Genetic material characterization ...................................................................... 66 3.1.3 Transformation ................................................................................................... 67 3.1.4 Transient Transfection ....................................................................................... 68 3.1.5 Stable transfection .............................................................................................. 68 3.1.6 Description of the Flp-In™ System ................................................................... 69 3.1.7 Cell line cloning and selection ........................................................................... 72 3.1.8 Lab scale fermentation ....................................................................................... 73 3.1.9 Cell banking ....................................................................................................... 73 3.2 Product characterization ............................................................................................ 74 3.2.1 SDS-PAGE ........................................................................................................ 74 3.2.2 Protein absorbance – NanoDrop ........................................................................ 75 3.2.3 Detection of product – Western blot .................................................................. 75 3.2.4 Identification of product – MALDI TOF MSMS .............................................. 77 3.2.5 Quantification of product ................................................................................... 80 3.3 Fermentation and downstream processing ................................................................ 83 3.3.1 Harvest ............................................................................................................... 83 4 3.3.2 Capture step – Anion Exchange chromatography ............................................. 83 3.3.3 Intermediate step – Hydrophobic interaction chromatography ......................... 84 3.4 Impurities characterization and cell stress ................................................................ 86 3.4.1 Zymography ....................................................................................................... 86 3.4.2 CHO HCP - AlphaLISA .................................................................................... 87 3.4.3 HCP identification and quantification via tandem MSMS ................................ 90 3.4.4 Lysosomal protein production pathway: TEM imaging .................................... 92 4 Chapter one ....................................................................................................................... 96 4.1 Introduction ............................................................................................................... 96 4.2 Plasmid generation .................................................................................................... 97 4.2.1 Cloning GAA into plasmid vectors.................................................................... 98 4.2.2 Transformation ................................................................................................... 99 4.2.3 Direction of insertion ....................................................................................... 100 4.2.4 Gene sequencing .............................................................................................. 103 4.3 Transfection into mammalian CHO ........................................................................ 106 4.3.1 Transient transfection....................................................................................... 107 4.3.2 Stable transfection ............................................................................................ 109 4.4 Cell line selection .................................................................................................... 112 4.4.1 Clone generation .............................................................................................. 112 4.4.2 Clone comparison ............................................................................................ 115 5 4.5 Chapter conclusions ................................................................................................ 117 5 Chapter two..................................................................................................................... 119 5.1 Introduction ............................................................................................................. 119 5.2 Target molecule size and presence. ......................................................................... 122 5.3 Target molecule identity.......................................................................................... 126 5.4 Target molecule quantification................................................................................ 130 5.4.1 Quantification by AlphaLISA .......................................................................... 130 5.4.2 Quantification by GAA diagnostic assay ......................................................... 137 5.5 Chapter conclusions ................................................................................................ 141 6 Chapter three ................................................................................................................... 143 6.1 Introduction ............................................................................................................. 143 6.2 Fermentation and harvest ........................................................................................ 144 6.3 Capture step– anion exchange chromatography...................................................... 144 6.3.1 Chromatogram ................................................................................................. 146 6.3.2 Fraction analysis .............................................................................................. 147 6.4 Intermediate purification – hydrophobic interaction chromatography ................... 152 6.4.1 HIC development strategy ..............................................................................
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