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Publié par | rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen |
Publié le | 01 janvier 2011 |
Nombre de lectures | 209 |
Langue | Deutsch |
Poids de l'ouvrage | 5 Mo |
Extrait
High level recombinant antibody production in Chinese hamster
ovary (CHO) cells and characterisation of the carcinoembryonic
antigen (CEA) specific human full-size IgG1 H10
Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der
RWTH Aachen University zur Erlangung des akademischen Grades einer
Doktorin der Naturwissenschaften genehmigte Dissertation
vorgelegt von
Diplom Biologin
Anne Verena Peuscher
aus Freiburg im Breisgau
Berichter: Universitätsprofessor Dr. rer. nat. Rainer Fischer
Universitätsprofessor Dr. rer. nat. Dr. rer. medic. Stefan Barth
Tag der mündlichen Prüfung: 8.4.2011
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.
Für meine Familie Eidesstattliche Erklärung:
Hiermit bestätige ich, die vorliegende Arbeit selbständig angefertigt zu haben und keine
anderen Hilfsmittel und Quellen als die im Text erwähnten verwendet zu haben.
Aachen, im April 2011
(Anne Peuscher)
Table of contents
I Introduction .................................................................................................. 1
I.1 Recombinant protein production in Chinese hamster ovary (CHO) cells ........ 1
I.1.1 Chinese hamster ovary cells ............................................................................... 2
I.1.2 MTX-mediated gene amplification .................................................................... 3
I.1.3 Generation of a monoclonal production cell line ............................................... 4
I.2 Biopharmaceutical monoclonal antibodies ....................................................... 5
I.2.1 Glycostructure dependent effector function 5
I.2.2 Glyco-modification of Fc linked oligosaccharides ............................................ 7
I.2.3 The carcinoembryonic antigen (CEA) as a tumor marker ................................. 8
I.2.4 Anti-CEA antibodies ........................................................................................ 10
I.3 Aim of the PhD thesis ..................................................................................... 12
II Material and methods ................................................................................ 15
II.1 Material ........................................................................................................... 15
II.1.1 Chemicals and consumables ............................................................................. 15
II.1.2 Enzymes and reaction kits 15
II.1.3 Antibodies, ligands and antigens ...................................................................... 16
II.1.4 Substrates ......................................................................................................... 17
II.1.5 Bacterial strain .................................................................................................. 17
II.1.6 Mammalian cell lines ....................................................................................... 17
II.1.7 Human tissue .................................................................................................... 18
II.1.8 Plasmids ........................................................................................................... 18
II.1.9 Oligonucleotides ............................................................................................... 19
II.1.10 Solutions, media and buffers ............................................................................ 19
II.1.11 Media and supplements for the cultivation and isolation of mammalian cells 19
II.1.12 Matrices and membranes .................................................................................. 21
II.1.13 Equipment ........................................................................................................ 21
II.1.14 Software ........................................................................................................... 22
II.2 Methods ........................................................................................................... 23
II.2.1 Recombinant DNA technology ........................................................................ 23
II.2.1.1 Cultivation of E. coli ............................................................................................... 23
II.2.1.2 Generation of electrocompetent E. coli................................................................... 23
II.2.1.3 Electrotransformation of E. coli .............................................................................. 23
II.2.1.4 Generation of glycerol stocks for long-term storage of E. coli strains ................... 24
II.2.1.5 Isolation of plasmid DNA from E. coli ................................................................... 24 Table of contents
II.2.1.6 Agarose gelelectrophoresis of DNA ....................................................................... 24
II.2.1.7 Polymerase Chain Reaction (PCR) ......................................................................... 25
II.2.1.8 Restriction enzyme digestion of DNA and cohesive end fill-in ............................. 26
ylation and ligation of restriction enzyme digested DNA ................... 26 II.2.1.9 Dephosphor
II.2.1.10 Dialysis of ligation samples for salt removal .......................................................... 27
II.2.1.11 Sequencing of plasmid DNA .................................................................................. 27
II.2.1.12 Phenol-chloroform-isoamyl alcohol (PCI) and ethanol precipitation ..................... 27
II.2.2 Mammalian cell culture methods ..................................................................... 28
II.2.2.1 Cultivation of mammalian cell cultures .................................................................. 28
II.2.2.2 Cryopreservation and thawing of mammalian cells ................................................ 28
II.2.2.3 Transfection and generation of stable transfected DG44 cells ................................ 28
II.2.2.4 MTX-mediated gene amplification ......................................................................... 29
II.2.2.5 Limiting dilution (LD) ............................................................................................ 29
II.2.2.6 Flow cytometric analysis and sorting...................................................................... 30
II.2.2.6.1 Flow cytometric analysis ................................................................................ 31
II.2.2.6.2 Fluorescence activated cell sorting (FACS) ................................................... 32
II.2.2.7 Procedure of whole cell extract preparation ........................................................... 32
II.2.2.8 Nomenclature of H10 producing DG44 cells ......................................................... 33
II.2.2.9 Separation of peripheral blood mononuclear cells (PBMC) from blood and
isolation of natural killer (NK) cells ........................................................................ 33
II.2.2.10 ADCC assay ............................................................................................................ 34
II.2.3 H10 purification and labeling ........................................................................... 35
II.2.3.1 Purification of H10 via Protein A affinity chromatography ................................... 35
II.2.3.2 Buffer exchange of protein solutions ...................................................................... 35
II.2.3.3 Conjugation of H10 with biotin or DyLight549 ..................................................... 36
II.2.4 Proteinchemical and immunological methods ................................................. 36
II.2.4.1 SDS-Polyacrylamide gelelectrophoresis (SDS-PAGE) and Coomassie staining ... 36
II.2.4.2 Immunoblot analysis ............................................................................................... 38
II.2.4.3 Enzyme linked immuno sorbent assay (ELISA) ..................................................... 38
II.2.4.4 Surface plasmon resonance (SPR) based quantification and analysis .................... 39
II.2.4.5 Immunofluorescence staining ................................................................................. 40
II.2.4.5.1 Immng of fixed cells ................................................... 40
II.2.4.5.2 Immng of tissue sections (immunohistochem