and Bivalent Antibody Derivatives

131 Pages·2013·5.84 MB·English

Cell Line Development for and Characterization of
Mono- and Bivalent Antibody Derivatives

Von der Fakultät Energie-, Verfahrens- und Biotechnik der
Universität Stuttgart zur Erlangung der Würde eines Doktors der
Naturwissenschaften (Dr. rer. nat.) genehmigte Abhandlung

Vorgelegt von
Verena Berger
aus Dresden

Hauptberichter: Prof. Dr. Roland Kontermann
Mitberichter: Prof. Dr. Klaus Pfizenmaier

Tag der mündlichen Prüfung: 12. Juli 2013

Institut für Zellbiologie und Immunologie der Universität Stuttgart 2013

I hereby declare that I performed the present thesis independently without further help or
other material than stated.

Verena Berger

Jülich, May 13, 2013

Cell Line Development for and Characterization of Contents
Mono- and Bivalent Antibody Derivatives

TABLE OF CONTENTS
Table of contents ........................................................................................................................ 3
Abbreviations ............................................................................................................................. 6
Summary .................................................................................................................................... 8
Zusammenfassung .................................................................................................................... 10
1 Introduction ...................................................................................................................... 12
1.1 Therapeutic Monoclonal Antibodies ......................................................................... 12
1.1.1 Antibody Structure and Function ....................................................................... 13
1.2 Antibody Engineering ................................................................................................ 14
1.2.1 Engineered Fc Function ..................................................................................... 15
1.3 Therapeutic Antibody Fragments .............................................................................. 15
1.4 Human Serum Albumin ............................................................................................. 18
1.5 Antibody-based therapeutics in TNF-related diseases .............................................. 19
1.6 Antagonistic Anti TNFR1-specific Antibody (ATROSAB) ..................................... 22
1.7 Immunogenicity ......................................................................................................... 23
1.8 Manufacturing of recombinant biotherapeutics ......................................................... 24
1.9 Aim of the thesis ........................................................................................................ 27
2 Material and Methods ....................................................................................................... 28
2.1 Material ...................................................................................................................... 28
2.1.1 Equipment .......................................................................................................... 28
2.1.2 Consumables ...................................................................................................... 29
2.1.3 Special Implements ............................................................................................ 30
2.1.4 Chemicals ........................................................................................................... 30
2.1.5 Reagents ............................................................................................................. 31
2.1.6 Buffers and solutions .......................................................................................... 31
2.1.7 Cell lines ............................................................................................................. 32
2.1.8 Vectors ............................................................................................................... 33
2.1.9 Bacteria strains ................................................................................................... 33
2.1.10 Kits, Markers ...................................................................................................... 33
2.1.11 Antibodies .......................................................................................................... 34
2.1.12 Enzymes ............................................................................................................. 34
2.1.13 Media & Supplements ........................................................................................ 35
2.2 Methods ..................................................................................................................... 35
2.2.1 Cloning ............................................................................................................... 35
2.2.1.1 Restriction digest ........................................................................................ 35
2.2.1.2 Isolation of DNA fragments ........................................................................ 35
2.2.1.3 Generation of blunt ends ............................................................................. 36
2.2.1.4 Dephosphorylation ...................................................................................... 36
2.2.1.5 Ligation ....................................................................................................... 36
2.2.1.6 Transformation ............................................................................................ 37
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Contents Cell Line Development for and Characterization of
Mono- and Bivalent Antibody Derivatives

2.2.1.7 Preparation of transfection grade plasmid DNA ......................................... 37
2.2.1.8 Cloning of ATROSAB ................................................................................ 37
2.2.1.9 Cloning of scFv-HSA ................................................................................. 38
2.2.1.10 Sequencing .................................................................................................. 38
2.2.2 Cell culture ......................................................................................................... 39
2.2.2.1 Cultivation cell lines 23-CHO-S and 14-CHO-S/CV063/25.004 ............... 39
2.2.2.2 Cultivation of cell lines HT1080, HeLa and MEF-TNFR1-Fas/MEF-
TNFR2-Fas ................................................................................................................... 39
2.2.3 Cell line development ......................................................................................... 39
2.2.3.1 Transfection ................................................................................................ 39
2.2.3.2 Selection of high producer clones - SEFEX ............................................... 40
2.2.3.3 Selection of high producers – CEMAX ...................................................... 41
2.2.3.4 Expansion of producer clones ..................................................................... 41
2.2.3.5 Test for cell specific productivity ............................................................... 41
2.2.3.6 Cryopreservation ......................................................................................... 42
2.2.4 Protein production .............................................................................................. 42
2.2.4.1 Purification of recombinant scFv from the periplasm ................................ 42
2.2.4.2 Research scale production of TNFR1 antagonists and hTNFR1 ................ 42
2.2.5 Protein purification ............................................................................................. 43
2.2.5.1 Protein A chromatography .......................................................................... 43
2.2.5.2 Purification by blue sepharose .................................................................... 43
2.2.5.3 Immobilized metal affinity chromatography (IMAC) ................................ 43
2.2.6 Biochemical characterization ............................................................................. 44
2.2.6.1 SDS-PAGE and Western Blot Analysis...................................................... 44
2.2.6.2 Nonequilibrium pH Gel Electrophoresis (NEPHGE) ................................. 44
2.2.6.3 Thermal stability ......................................................................................... 44
2.2.6.4 Size Exclusion Chromatography (SEC) ...................................................... 45
2.2.6.5 Cation exchange chromatography (CEX) ................................................... 45
2.2.7 Functional characterization ................................................................................ 46
2.2.7.1 Serum Stability ............................................................................................ 46
2.2.7.2 Affinity measurement ................................................................................. 46
2.2.7.3 Enzyme linked immune absorbent assay (ELISA) ..................................... 46
2.2.7.4 Multiplex Assay .......................................................................................... 47
2.2.7.5 Flow Cytometry .......................................................................................... 47
2.2.7.6 Cytotoxicity ................................................................................................. 47
2.2.7.7 Human whole blood assay .......................................................................... 48
2.2.7.8 IL-6 and IL-8 assay ..................................................................................... 48
3 Results .............................................................................................................................. 49
3.1 Development of research cell lines for three different structure variants of IZI06.1 49
3.1.1 Cloning of the vector constructs ......................................................................... 50
3.1.2 Development of research cell lines .................................................................... 50
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Cell Line Development for and Characterization of Contents
Mono- and Bivalent Antibody Derivatives

3.1.3 Characterization of clones .................................................................................. 51
3.2 Evaluation of monovalent TNFR1 inhibitor scFv-HSA ............................................ 58
3.2.1 Production of scFv-HSA fusion protein ............................................................. 58
3.2.2 Biochemical and functional characterization of the scFv-HSA fusion protein .. 59
3.2.2.1 Thermal stability ......................................................................................... 59
3.2.2.2 Binding specificity and affinity .................................................................. 60
3.2.2.3 Functional inhibition of TNF induced IL-6 and IL-8 release ..................... 61
3.2.2.4 Pharmacokinetics ........................................................................................ 63
3.3 Development of a regulatory compliant production cell line for ATROSAB ........... 63
3.3.1 Vector construction pCV107 .............................................................................. 63
3.3.2 Cell line generation 23-CHO-S/CV107/K20 ..................................................... 66
3.3.2.1 Characterization of potential producer clones ............................................ 73
3.3.2.2 Stability of protein expression .................................................................... 75
3.3.3 Cell line generation 23-CHO-S/CV107/K20-3/CV108/K35-1 and /K35-2 ....... 77
3.3.3.1 Characterization of potential producer clones ............................................ 82
3.3.3.2 Comparison of ATROSAB expressing cell lines 23-CHO-S/CV107/K20-3
and 23-CHO-S/CV107/K20-3/CV108/K35-2 .............................................................. 87
3.4 Establishment of a an assay for the ex-vivo proof of ATROSAB action in clinical
blood samples ....................................................................................................................... 92
3.5 Tables ......................................................................................................................... 96
4 Discussion ........................................................................................................................ 98
4.1 Generation research cell lines for the three structure variants ................................... 98
4.2 Impact of composition, size and valency on effective huTNFR1 antagonism ........ 103
4.3 Regulatory compliant cell line generation for ATROSAB ..................................... 105
4.3.1 Limiting Dilution .............................................................................................. 106
4.3.2 Evaluation of cell line performance ................................................................. 107
4.3.3 Growth and expression stability ....................................................................... 108
4.3.4 Biochemical and functional comparison of ATROSAB produced by K20-3 and
K35-2 .......................................................................................................................... 109
4.3.5 Establishment of a test for assessing biological functionality of ATROSAB .. 109
5 References ...................................................................................................................... 112
6 Acknowledgements ........................................................................................................ 126
7 Curriculum vitae ............................................................................................................. 127

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Abbreviations Cell Line Development for and Characterization of
Mono- and Bivalent Antibody Derivatives

ABBREVIATIONS
(HP-) SEC High pressure size exclusion EDTA Ethylenediaminetetraacetate
chromatography ELISA Enzyme-linked immuno-
(m) Ab (monoclonal) Antibody sorbent assay
(R) WCB (Research) working cell Fab Fragment antigen binding
bank Fc Fragment crystallizable
ADA Anti drug antibody FcRn Neonatal Fc-Receptor
ADCC Antibody dependent FCS/FBS Fetal calf serum
cytotoxicity Fetal bovine serum
AUC Area under the curve FDA Food and Drug
BCA Bicinchoninic acid assay Administration
BGH Bovine growth hormone FPLC Fast protein liquid
bp Base pairs chromatography
BSA Bovine serum albumin G418 Geneticin
CCK-8 Cell Counting Kit 8 GMP Good Manufacturing
CDC Complement dependent Practice
cytotoxicity GOI Gene of interest
CDR Complementarity GS Glutamin synthetase
determining region h hour
CDS Coding sequence hu human
CEX Cation exchange H SO Sulfuric acid
2 4
chromatography HACA Human anti chimeric
CH Constant heavy chain antibodies
CHO Chinese hamster ovary HAHA Human anti human
CL Constant light chain antibodies
CMV Cytomeglalovirus HAMA Human anti mouse
CRD Cystein rich domain antibodies
CSP Cell specific productivity HCl Hydrogen hhloride
CV Column volume HCP Host cell protein
CV/AV Celonic Vector / Order hEF1 Human elongation factor 1
Vector HES Hydroxyethyl starch
d day HPLC High pressure liquid
DHFR Dihydrofolate reductase chromatography
DMSO Dimethyl sulfoxide HRP Horse raddish peroxidase
DNA Deoxyribonucleic acid HSA Human serum albumin
dNTP Deoxyribonucleotide IBD Inflammatory Bowel
dO Dissolved oxygen Disease
DTT Dithiothreitol IC Half maximal inhibitory
50
EC Half maximal effective concentration
50
concentration Ig Immunoglobuline
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Cell Line Development for and Characterization of Abbreviations
Mono- and Bivalent Antibody Derivatives

IL Interleukin PK Pharmacokinetics
IMAC Immobilized metal ion PLAD Pre-ligand assembly domain
affinity chromatography POD Peroxidase
IMDM Immune mediated PSB Primary seed bank
inflammatory disease QCM Quartz crystal microbalance
IPTG Isopropyl β-D-1- r Recombinant
thiogalactopyranoside RA Rheumatoid arthritis
I-SceI Meganuclease RCME Recombinase mediated
IVCD Integrated viable cell cassette exchange
density rpm Rounds per minute
kcps kilocycle per second RT Room temperature
(kilohertz) scFv Single chain fragment
K Dissociation constant variable
D
LB Luria broth SDS-PAGE Sodium dodecyl sulfate
LD Limiting dilution polyacrylamide gel
LD 90 % Lethal dosis Electrophoresis
90
LPS Lipopolysaccharide SOC/SOB Superoptimal broth
MCS Multiple cloning site TBE Tris borate EDTA buffer
MEF Mouse embryonic TBS Tris buffered saline
fibroblasts TCA Trichloroacetic acid
MgSO Magnesium sulfate tcd Total cell density
4
MHC Major histocompatibility TE Tris EDTA buffer
complex TLR Toll-like receptor
min Minutes TMB 3,3’,5,5’-Tetramethyl-
MS Multiple sclerosis benzidine
MTX Methotrexate TNF Tumor necrosis factor
MW Molecular weight TNFR Tumor necrosis factor
NaOH Sodium hydroxide receptor
NaPO Sodium phosphate TRAPS TNF receptor associated
4
NEPHGE Nonequilibrium pH gel periodic syndrome
electrophoresis UF/DF Ultrafiltration / Diafiltration
NF Nucleofection UV Ultraviolet light
OD Optical density vcd Viable cell density
pA Polyadenylation signal VH Variable heavy chain
PBS Phosphate buffered saline VL Variable light chain
PDT Population doubling time
PEG Polyethylenglycol

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Summary Cell Line Development for and Characterization of
Mono- and Bivalent Antibody Derivatives

SUMMARY
Immune mediated inflammatory diseases (IMDM) can affect a variety of tissues and organs
causing a broad spectrum of symptoms. Yet, rheumatoid arthritis, multiple sclerosis, psoriasis
or inflammatory bowel disease share mechanistic similarities. A major factor in the
development is the imbalance of tumor necrosis factor (TNF) regulation. With a prevalence of
5-7 % in the western society, a great interest exists in the development of an effective therapy.
Current treatment involves primarily corticosteroids and non-biologic disease-modifying
antirheumatic drugs (DMARDs) like methotrexate (MTX). The fast progression made in the
development of biopharmaceutics, especially of antibody based therapeutics allowed for a
more specific treatment. TNF-related diseases are tried to tackle by intervention in the TNF
signaling. To date, five such therapeutics are approved by the US Food and Drug
Administration (FDA). They are aiming at the neutralization of TNF or lymphotoxin alpha
(LTa ), thereby reducing exaggerated TNF signaling and curtailing inflammation. However,
neutralization of TNF facilitates opportunistic infections and several warnings have been
issued by the FDA. In 1990, an antibody directed specifically against TNFR1 was discovered,
when mice were immunized with human TNFR1. A humanized variant was generated in
2008, paving the way for the use of this antibody as a therapeutic in humans.

The present study describes the development of chinese hamster ovary (CHO) cell lines
expressing three different variants of the humanized TNFR1 specific antibody IZI06.1, a full
length antibody and two human serum albumin fusion proteins (scFv-HSA and Fab-HSA).
Cell lines were generated by site directed integration of the GOI into the CHO host genome in
order to investigate the influence of the individual construct on growth performance and
producibility. Cell lines expressing the IgG1 and the scFv-HSA perfomed similarly, showing
productivities of 5-10 pg/c/d. Both proteins assembled properly and could be purified to
satisfactory purity. These constructs were found to be applicable for the generation of a
regulatory compliant cell line. The Fab-HSA revealed multiple issues in terms of productivity
and purification and was therefore excluded from further investigation.

The monovalent fusion protein scFv-HSA and full length antibody (termed ATROSAB) were
investigated in more detail to determine the impact of variables like composition, size and
valency on their biochemical and functional properties. Reduced size of the fusion protein
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