Generation of high expressing CHO cell lines for the production of recombinant antibodies using ...

179 Pages·2012·5.27 MB·English

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
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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 vector 726 88
Fig. 46: Fed-batch experiment with selected Master 2 cell lines 90
Fig. 47: Overview of used ER stress reporter and control constructs (containing eGFP) 92
Fig. 48: Overview of used ER stress reporter and control constructs (containing d2eGFP) 93
Fig. 49: Overview of used ER stress reporter construct (containing an artificial variant of
d2eGFP) 94
Fig. 50: Schematic representation of the cassette exchange procedure 94
Fig. 51: FACS analysis and single cell sorting of Master 2 and Mock cell lines after RMCE with
ER stress reporter or control constructs 98
v
List of figures
Fig. 52: PCR analysis of Mock and Master 2 cell clones exchanged with ER stress reporter or
control constructs 100
Fig. 53: FACS analysis of exchanged cell pools derived from Mock and Master 2 cell lines 102
Fig. 54: FACS analysis of Mock and Master 2 cell clones authentically exchanged with ER
stress reporter or control constructs 103
Fig. 55: FACS analysis of Mock and Master 2 Reporter cell lines treated with BFA or CSN 105
Fig. 56: Schematic representation of the generation of the trunc GRP78 Reporter cell line stably
expressing a recombinant antibody 107
Fig. 57: Schematic representation of vector 527 107
Fig. 58: FACS analysis of trunc GRP78 Reporter cell line 108
Fig. 59: Schematic representation of vector 314 108
Fig. 60: Fed-batch Experiments with selected production clones derived from the trunc GRP78
Reporter cell line 110
Fig. 61: Correlation between fed-batch performance (including antibody productivity) and GFP
fluorescence in production clones derived from the trunc GRP78 Reporter cell line 111
Fig. 62: FACS analysis of selected production clones derived from the tunc GRP78 Reporter
cell line after treatment with BFA or CSN 113
Fig. 63: Real time RT-PCR analysis of selected production clones derived from the trunc
GRP78 Reporter cell line after treatment with BFA 114
Fig. 64: FACS analysis of Mock Reporter and Master 2 Reporter cell lines 143
Fig. 65: FACS analysis of selected production clones derived from the tunc GRP78 Reporter
cell line after treatment with BFA or CSN 145
vi