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Generation of High Expressing CHO Cell Lines for The Generation of high expressing CHO cell lines for the production of recombinant antibodies using optimized signal peptides and a novel ER stress based selection system Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Lars Kober aus Riesa 1. Referent: apl. Professor Dr. Jürgen Bode 2. Referent: Professor Dr. Stefan Dübel Eingereicht am: 02.02.2012 Mündliche Prüfung (Disputation) am: 15.06.2012 Druckjahr 2012 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Kober L, Zehe C, Bode J. 2012. Development of a novel ER stress based selection system for the isolation of highly productive clones. (ahead of print) Kober L, Zehe C, Bode J. 2012. Optimized signal peptides for the development of high expressing CHO cell lines. (in submission) Quote „The fact that an opinion has been widely held is no evidence whatever it is not utterly absurd” Bertrand Russell (1872-1970) Contents Contents page 1. Zusammenfassung.................................................................................................................. 1 2. Abstract ................................................................................................................................... 3 3. Introduction.............................................................................................................................. 5 3.1. High-level production of recombinant proteins in CHO cells ....................................... 5 3.2. Protein secretion and signal peptides .......................................................................... 7 3.3. Mammalian ER stress................................................................................................... 9 4. Aim of the thesis .................................................................................................................... 13 5. Materials and methods .......................................................................................................... 14 5.1. Materials...................................................................................................................... 14 5.2. Molecular biological methods ..................................................................................... 24 5.3. Cell biological methods ............................................................................................... 50 5.4. Analytical and biochemical methods .......................................................................... 57 6. Results................................................................................................................................... 62 6.1. Increase of antibody secretion by signal peptide optimization................................... 62 6.2. General outline: Development of an alternative selection system for high producing clones .................................................................................................................................... 77 6.3. Analyzing the influence of antibody expression on ER stress ................................... 81 6.4. Generation and analysis of Master 1 cell lines containing an RMCE acceptor construct (RMCE target) ....................................................................................................... 84 6.5. Generation and analysis of Mock and Master 2 cell lines.......................................... 88 6.6. Generation of ER stress reporter cell lines................................................................. 91 6.7. Evaluating the influence of antibody expression on ER stress reporter constructs 101 i Contents 6.8. Impact of BFA or CSN treatment on ER stress reporter constructs ........................ 104 6.9. Establishment of a novel ER stress based selection system for the isolation of high- producing clones ................................................................................................................. 106 7. Discussion ........................................................................................................................... 115 7.1. Increase of antibody secretion by signal peptide optimization................................. 115 7.2. Development of an alternative selection system for high producing clones ............ 118 8. Outlook ................................................................................................................................ 124 9. Appendix.............................................................................................................................. 125 9.1. FACS analysis of Mock Reporter and Master 2 Reporter cell lines......................... 125 9.2. Abbreviations ............................................................................................................ 146 10. References ....................................................................................................................... 150 10.1. Literature ............................................................................................................... 150 10.2. Patents .................................................................................................................. 164 11. Acknowledgements .......................................................................................................... 165 ii List of figures List of figures page Fig. 1: Schematic representation of ER stress pathways 11 Fig. 2: Nucleotide sequence of the first two codons of the immunoglobulin heavy chain 25 Fig. 3: Nucleotide sequence of the first two codons of the immunoglobulin light chain 25 Fig. 4: Schematic representation of the cloning procedure for first generation expression vectors 26 Fig. 5: Schematic representation of the cloning procedure for second generation expression vectors 27 Fig. 6: Schematic representation of the cloning procedure for third generation expression vectors 28 Fig. 7: Nucleotide sequence of the BbsI/BbsI cloning site in front of the immunoglobulin heavy chain 28 Fig. 8: Nucleotide sequence of the BsmBI/BsmBI cloning site in front of the immunoglobulin light chain 28 Fig. 9: Schematic representation of vector 1188 (SV40-eGFP control) 29 Fig. 10: Nucleotide sequence upstream of the eGFP reporter gene of vector 1188 (SV40-eGFP control) 29 Fig. 11: Nucleotide sequence upstream of the eGFP reporter gene of vector 1192 (3 x ERSE I reporter) 30 Fig. 12: Nucleotide sequence upstream of the eGFP reporter gene of vector 1193 (3 x ERSE II reporter) 30 Fig. 13: Nucleotide sequence upstream of the eGFP reporter gene of vector 1194 (3 x UPRE reporter) 31 Fig. 14: Nucleotide sequence upstream of the eGFP reporter gene of vector 1195 (3 x AARE reporter) 31 Fig. 15: Schematic representation of vector 917 (SV40-d2eGFP control) 32 iii List of figures Fig. 16: Nucleotide sequence upstream of the d2eGFP reporter gene of vector 917 (SV40- d2eGFP control) 32 Fig. 17: Nucleotide sequence upstream of the d2eGFP reporter gene of vector 1101 (CALR reporter) 33 Fig. 18: Nucleotide sequence upstream of the d2eGFP reporter gene of vector 1103 (GRP78 reporter) 34 Fig. 19: Nucleotide sequence upstream of the d2eGFP reporter gene of vector 1104 (GRP94 reporter) 35 Fig. 20: Nucleotide sequence of the XBP1 intron reporter gene of vector 1023 (Intron Reporter) 36 Fig. 21: Schematic representation of the cloning procedure of vector 527 37 Fig. 22: Nucleotide sequence upstream of the eGFP reporter gene of vector 527 37 Fig. 23: Schematic representation of vector 673 38 Fig. 24: Schematic representation of vector 957 39 Fig. 25: Schematic representation of vector 1071 40 Fig. 26: Schematic representation of vector 1096 41 Fig. 27: Screening of different natural and artificial signal peptides in transiently transfected CHO K1 cells 65 Fig. 28: Evaluation of selected signal peptides in transiently transfected CHO K1 cells 66 Fig. 29: Secondary mRNA structure of signal peptide E and E8 69 Fig. 30: Analysis of natural and mutated signal peptides in transiently transfected CHO K1 cells 70 Fig 31: Evaluation of selected natural and mutated signal peptides in transiently transfected CHO K1 cells 71 Fig. 32: Cell specific productivities of mini-pools used for fed-batch experiments 74 Fig. 33: Fed-batch experiments with selected mini-pools 75 iv List of figures Fig. 34: Fed-batch experiments with selected mini-pools 76 Fig. 35: Schematic representation of the novel selection system based on ER stress (exemplified shown with GRP78 promoter) 77 Fig. 36: General outline of the generation of Master 1 Master 2A and Mock cell lines and their conversion to ER stress reporter cell lines 79 Fig. 37: General outline of the conversion of Master 2A and Mock cell lines in control cell lines 80 Fig. 38: Correlation between antibody productivity and mRNA levels of ER stress factors in selected production clones 83 Fig. 39: Schematic representation of vector 957 84 Fig. 40: Single cell sorting after transfection with vector 957 84 Fig. 41: FACS analysis of Master 1 cell clones containing a DsRed RMCE acceptor construct 85 Fig. 42: Schematic representation of RMCE procedure 85 Fig. 43: FACS analysis of Master 1 cell clones before and after RMCE 86 Fig. 44: Schematic representation of vector 1096 88 Fig. 45: Schematic representation of
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