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BAHAGIAN A – Pengesahan Kerjasama*
Adalah disahkan bahawa projek penyelidikan tesis ini telah dilaksanakan melalui
kerjasama antara _______________________ dengan _______________________
Disahkan oleh:
Tandatangan : Tarikh :
Nama :
Jawatan :
(Cop rasmi)
* Jika penyediaan tesis/projek melibatkan kerjasama.
BAHAGIAN B – Untuk Kegunaan Pejabat Sekolah Pengajian Siswazah
Tesis ini telah diperiksa dan diakui oleh:
Nama dan Alamat Pemeriksa Luar : Prof. Dr. Arbakariya Ariff
Department of Bioprocess Technology,
Faculty of Biotechnology & Biomolecular
Sciences, UPM 43400 Serdang, Selangor.
Nama dan Alamat Pemeriksa Dalam : Prof. Madya Dr. Firdausi Razali
Department of Bioprocess Engineering,
Faculty of Chemical & Natural Resources
Engineering (FKKKSA), UTM, 81310
UTM, Skudai, Johor.
Nama Penyelia Lain (jika ada) :
Disahkan oleh Timbalan Pendaftar di SPS:
Tandatangan : Tarikh :
Nama :
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CHROMATOGRAPHIC PURIFICATION STRATEGIES FOR RECOMBINANT
HUMAN TRANSFERRIN FROM SPODOPTERA FRUGIPERDA
WEE CHEN CHEN
A thesis submitted in fulfilment of the
requirements for the award of the degree of
Master of Engineering (Bioprocess)
Faculty of Chemical and Natural Resources Engineering
Universiti Teknologi Malaysia
JUNE 2008
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To my beloved grandparents, parents and brothers
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ACKNOWLEDGEMENT
In this long research journey, I receive all kind of guidance and support,
technically, financially and also spiritually. Thanks to everyone and the institution
for making this work possible. Special thanks are due to my supervisor, PM. Dr
Azila Abdul Aziz and co-supervisor, Dr. Badarulhisam Abdul Rahman for the
opportunity to be involved in this interesting project, their professional advice and
their encouragement in the effort to complete this research. I appreciate the given
opportunity to have a closer insight into biomanufacturing industry. Thanks also to
Prof. Dr. Michael J. Betenbaugh of Johns Hopkins University, USA for providing
recombinant baculoviruses.
I would like to express gratitude to all Bioprocess Department laboratory staff
especially Puan Siti Zalita, Encik Muhammad, Encik Malek and Encik Yaakop. I
also would like to thank all of the staff of Research Manage Center and Faculty of
Chemical and Natural Resources Engineering especially Cik Yun and Pn Naza.
They have been very helpful. I am also feel gratitude to have a group of kind
labmates and friends. Thanks to Dr. Taher, Wei Ney, Clarence, Hafiz, Kamalesh and
Kian Mou for their knowledge sharing. “Fui Ling, Melissa, Lee Yu and Seat Yee,
thanks for your company and motivation through out this long run”. Not to forget,
thanks to all the teachers and lecturers who had taught me all the basic knowledge.
My deepest appreciation would be dedicated to my sweet family members.
Their patience, consideration, encouragement, consistent support, recognition and
invaluable love make me strong and proud. Thank you.
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ABSTRACT
Insect cell-baculovirus system is an excellent artificial system for the
production of recombinant glycoprotein despite its glycosylation deficiencies. In this
study, laboratory scale production of recombinant human transferrin (rhTf) from
insect cell-BEVS was conducted and chromatographic purification strategies were
employed to obtain rhTf in high yield and high recovery. Research was started with
the amplification of recombinant baculovirus, using low multiplicity of infection
(MOI). Virus stock in a 1.2 x 109 pfu/ml infected suspension culture of Spodoptera
frugiperda (Sf9) at 15 MOI had produced 31µg/ml of rhTf. To purify the rhTf,
hydrophobic interaction chromatography, dialysis and ion exchange chromatography
were performed. For hydrophobic interaction chromatography, elution strategy,
flowrate and rhTf loading capacity of phenyl sepharose were optimized. By loading
38µg rhTf/ml of gel, employing step elution with 50% 1.2M (NH4)2SO4/0.4M
Na3C6H5O7, pH6 (buffer A) and 25% buffer A and flowrate at 1ml/min, 74.6% of
rhTf had been recovered from phenyl sepharose. For ion exchange chromatography,
batch purification in reduced size was used to select suitable anion exchange matrix,
suitable pH of equilibration buffer and concentration of equilibration buffer. 20mM
Tris/HCl buffer, pH8.5 and gradient elution with the increase of of 5mM NaCl/CV
succeeded in giving pure rhTf with 52.5% recovery from Q-sepharose. The overall
recovery of pure rhTf was 34% with 200 purification fold. A brief glycan
characterization of the recovered pure rhTf was performed for a better understanding
of the glycosylation feature of this protein expressed using optimized medium from
BEVS. The carbohydrate component of the purified rhTf was determined. The
purified rhTf was hydrolyzed and the release sugar was labeled with 1-Phenyl-3-
Methyl-5-Pyrazolone (PMP) before analysis with High performance Liquid
Chromatography (HPLC). The molar fractions of Man, GlcNAc and Gal of rhTf
were 3.78, 1.69 and 0.93, respectively.
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ABSTRAK
Sistem pengekspresan sel serangga-bakulovirus merupakan sistem pilihan
yang baik untuk menghasilkan rekombinan glikoprotein meskipun kekurangan
glikosilasi. Penghasilan produksi skala makmal mendapat rekombinan human
transferrin (rhTf) dari sistem sel serangga-bakulovirus dan strategi purifikasi jenis
kromatografi telah dijalankan untuk mendapatkan rhTf yang tulen dan perolehan
yang tinggi. Kajian bermula dengan peningkatan kuantiti rekombinan bakulovirus
dari gandaan jangkitan (MOI) yang rendah. Stok virus dalam 1.2 x 109 pfu/ml
menjangkiti kultur ampaian sel Spodoptera frugiperda (Sf9) dengan 15 MOI telah
menghasilkan 31µg/ml rhTf. Dalam proses purifikasi, kromatografi saling tindak
hidrofobik, dialisis dan kromatografi penukaran ion telah dijalankan. Bagi
kromatografi saling tindak hidrofobik, strategi elusi, kelajuan dan kapasiti muatan
rhTf ke atas phenyl sepharose telah dioptimumkan. Penggunaan muatan 38µg
rhTf/ml gel dengan elusi berperingkat menggunakan 50% 1.2M (NH4)2SO4/0.4M
Na3C6H5O7, pH6 (larutan penimbal A) and 25% larutan penimbal A dan kelajuan
pada 1ml/min berjaya memperoleh 74.6% rhTf daripada phenyl sepharose. Bagi
kromatografi penukaran ion, purifikasi dalam saiz kecil telah digunakan untuk
memilih matrik penukar ion, pH larutan penimbal pada fasa keseimbangan dan
kepekatan larutan penimbal pada fasa keseimbangan. 20mM Tris/HCl larutan
penimbal, pH8.5 and elusi cerun dengan peningkatan 5mM NaCl/CV berjaya
menghasilkan rhTf tulen dengan 52.5% perolehan daripada Q-sepharose. Perolehan
rhTf tulen secara keseluruhan ialah 34% dengan 200 lipat purifikasi. Pencirian
glikan secara kasar telah dijalankan ke atas rhTf tulen untuk mendapat pemahaman
tentang ciri-ciri glikosilasi bagi protein ini yang diekspresikan dengan sistem
pengekspresan sel serangga-bakulovirus dan media optimum. Komposisi
karbohidrat untuk rhTf tulen telah dikenalpasti. rhTf yang tulen telah dihidrolisis.
Gula telah dilepaskan, dan dilabelkan dengan 1-Phenyl-3-Methyl-5-Pyrazolone
(PMP) sebelum dianalisis dengan menggunakan kromatografi cecair prestasi tinggi
(HPLC). Nilai fraksi molar Man, GlcNAc and Gal daripada rhTf ialah 3.78, 1.69 and
0.93.
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TABLE OF CONTENTS
CHAPTER TITLE PAGE
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENTS iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENTS vii
LIST OF TABLES xii
LIST OF FIGURES xiii
LIST OF SYMBOLS/ ABBREVIATIONS xvi
1 INTRODUCTION 1
1.1 Preface 1
1.2 Objectives 6
1.2 Scopes of Research 6
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2 LITERATURE REVIEW 7
2.1 Recombinant Protein Expression System 7
2.2 Insect Cell Baculovirus Expression System 11
2.2.1 Insect cell 11
2.2.2 Baculoviruses 11
2.2.2.1 Invivo and Invitro Replication 13
2.2.2.2 Recombination 15
2.3 Glycosylation 17
2.3.1 N-Glycosylation and O-Glycosylation 17
2.3.2 Glyscosylation Pathway 20
2.3.2.1 Glycosylation Pathway in Insect Cell 21
2.3.3 Model Protein- Transferrin 23
2.3.3.1 Recombinant Human Transferrin 27
2.4 Analysis Method 28
2.4.1 Bicinchoninic Acid (BCA) Assay 28
2.4.2 Enzyme Linked Immunosorbent Assay (ELISA) 29
2.4.3 Sodium Dodecyl Sulfate -Polyacrylamide Gel
Electrophoresis (SDS-PAGE) 31
2.4.4 Western Blot 32
2.4.5 Glucose, Lactic Acid and Glutamine Analyzer 33
2.4.6 Carbohydrate Analysis Using High
Performance Liquid Chromatography (HPLC) 34
2.4.6.1 Hydrolysis 34
2.4.6.2 1-Phenyl-3-Methyl-5-Pyrazolone (PMP)
Derivative of Sugar 34
2.4.6.3 Reverse Phase-HPLC 35
2.5 Purification of Transferrin 36
2.5.1 Hydrophobic Interaction Chromatography
(HIC) 37
2.5.1.1 Factors Affecting HIC 39
2.5.2 Ion Exchange Chromatography 43
2.5.2.1 Factor Affecting IEX 44
2.5.3 Optimization Method in Process
Chromatography 48
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2.6 Summary of Literature Review 49
3 MATERIALS AND METHODS 51
3.1 Materials 51
3.1.1 Cell lines and Recombinant Baculovirus 51
3.1.2 Equipments 51
3.1.3 Chemicals 52
3.2 Spodoptera frugiperda (Sf-9) Cells Culture 53
3.2.1 Cells Thawing 53
3.2.2 Cells Count 54
3.2.3 Adapting Serum Contain Culture to Serum Free
Culture 55
3.2.4 Adapting Monolayer Cells to Suspension
Culture 55
3.2.5 Maintaining Suspension Culture 56
3.2.6 Preparation of Optimized Medium 56
3.2.7 Adapting Suspension Culture in SFM900II to
Optimized Medium 57
3.2.8 Cells Freezing 57
3.3 Recombinant Baculovirus 58
3.3.1 Generating Pure Recombinant Virus Stock 58
3.3.2 Amplification of Virus Stock 58
3.3.3 Optimization of rhTf Expression 59
3.3.4 Virus Titration (End-Point Dilution) 59
3.4 Recombinant Human Transferrin Detection 60
3.4.1 Enzyme Linked Immunosorbent Assay
(ELISA) 60
3.4.2 Sodium Dodecyl Sulfate -Polyacrylamide Gel
Electrophoresis 62
3.4.2.1 Silver Staining 63
A
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3.4.2.2 Coomassie Blue Staining 63
3.4.3 Western Blot 64
3.5 Characterization of Nutrient Consumption and
Substances Release 65
3.5.1 Analysis of Glucose, Lactic Acid and
Glutamine 65
3.5.2 Ammonia Test 66
3.6 Protein Assay 67
3.6.1 Bicinchoninic Acid (BCA) Assay 67
3.7 Purification 68
3.7.1 Hydropbobic Interaction Chromatography 68
3.7.2 Dialysis 69
3.7.3 Initial Screening Step of IEX using Batch
Purification in Reduced Volume 70
3.7.4 Ion Exchange Chromatography 71
3.8 Monosaccharide Composition Analysis of rhTf by
HPLC 72
3.8.1 Preparation of Apotransferrin, rhTf, Standard
Monosccharides 72
3.8.2 Hydrolysis 73
3.8.3 Pre-column Derivatization 73
3.8.4 HPLC Analysis 74
4 RESULTS AND DISCUSSION 75
4.1 Expression of rhTf 75
4.1.1 Growth Profile of Infected Virus 75
4.1.2 Time Course Expression Profile of rhTf 80
4.2 Purification 83
4.2.1 Profile of Sample Elution from Hydrophobic
Interaction Chromatography 85
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4.2.2 Optimization of Hydrophobic Interaction
Chromatography 86
4.2.2.1 Optimization of Elution Method 86
4.2.2.2 Optimization of Elution Flowrate 89
4.2.2.3 Optimization of rhTf Loading Capacity 92
4.2.3 Initial Screening Step of IEX Using Batch
Purification in Reduced Volume 95
4.2.4 Anion Exchange Chromatography 98
4.2.4.1 Maximizing The Selectivity of Anion
Exchange Chromatography 98
4.2.5 Characterization of rhTf Purification 100
4.3 Characterization of The Carbohydrate Composition of
rhTf 104
5 CONCLUSIONS 108
5.1 Conclusions 108
5.2 Recommendations 110
REFERENCES 112
APENDICES 137
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LIST OF TABLES
TABLE NO. TITLE PAGE
1.1 Comparison of pharmaceutical expression system
(Elbehri, 2005) 3
2.1 Characterization of selected host systems for protein
production from recombinant DNA (Shuler and Kargi, 2002) 10
2.2 Posttranslational processing and yield of the protein product in
various expression systems (cited from Luckow and Summers,
1988) 10
2.3 Selected private company with the protein engineering
platform 26
2.4 Functional groups used on ion exchangers 46
2.5 Capacity data for sepharose fast flow ion exchangers 47
2.6 Characteristics of Q, SP, DEAE and CM Sepharose Fast Flow 47
3.1 Culture volume for different flask size 56
3.2 Specification of YSI calibrator 65
3.3 Applied Condition for different study factors 71
4.1 Summary of the characteristic of small scale production of rhTf 83
4.2 Optimization of step-wise elution method for achieving higher
recovery of rhTf 87
4.3 Optimization of elution flowrate 90
4.4 Optimization of rhTf loading capacity 93
4.5 Summary of the characteristic of purification of rhTf 101
4.6 Carbohydrate Composition Analysis of Glycoprotein 107
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LIST OF FIGURES
FIGURE NO. TITLE PAGE
1.1 Worldwide sales forecast for protein drugs, 2006 and 2011
(Talukder, 2007) 2
1.2 Strength and weaknesses of various expression systems
(Cox, 2004) 4
2.1 Electron micrographs and schematic of baculoviruses 12
2.2 Structural compositions of the two baculovirus phenotypes,
budded virus (BV), and the polyhedron derived virus (PDV) 12
2.3 The baculovirus life cycle in vivo and in vitro 14
2.4 Construction of baculovirus expression vectors 16
2.5 Structure of the N-glycosidic bond and O-glycosidic bond
found in glycoproteins. 18
2.6 Structure of the different types of oligosccharidic chains of
N-glycoproteins 19
2.7 Pathway for generation of the dolichol-linked oligosaccharide donor
for protein N-glycosylation 21
2.8 Protein N-glycosylation pathways in insect and mammalian
cells 22
2.9 A ribbon diagram of a diferric rabbit serum transferrin
molecule 25
2.10 Reaction schematic for BCA assay 29
2.11 Schematic represents the a) Direct Sandwich ELISA; b)
Indirect ELISA; c) Sandwich ELISA; d) Competition ELISA 30
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2.12 SDS-PAGE 31
2.13 Immobilized enzyme biosensor of YSI 33
2.14 Hydrolysis time course of bovine fetuin 34
2.15 Derivatization with pyrazolone derivatives 35
2.16 Different hydrophobic ligands coupled to cross-linked
agarose matrices 40
2.17 The Hofmeister series on the effect of some anions and
cations in precipitating proteins 41
2.18 Relative effects of some salts on the molal surface tension of
water 41
2.19 Effect of pH on protein at different net charge 44
2.20 Ion exchanger types 45
3.1 Schematic representative of the procedures employed for
virus titer-end point dilution 60
3.2 Schematic representative of the procedures used in ELISA
method 61
3.3 Schematic representation of the BCA protein assay 67
3.4 Schematic diagram of the dialysis procedure 70
3.5 Schematic diagram of the set up of the chromatography
equipment. 72
4.1 Photography of control and infected culture 76
4.2 Growth Characteristics of sf9 during rhTf virus propagation 78
4.3 Growth Characteristic of sf9 during rhTf production in
optimized suspension culture 79
4.4 The profile of glucose, glutamine consumption and lactate
formation in supernatant post infection 80
4.5 rhTf production profile in supernatant 81
4.6 Characterization of the rhTf production profile of infected
Sf9, using 9%, Coomassie blue staining, SDS-PAGE 82
4.7 Characterization of the rhTf production profile of infected
Sf9, using Western Blot 82
4.8 Steps and gradient elutions of rhTf from HIC column 85
4.9 HIC chromatograms for the optimization of elution method 88
4.10 HIC chromatograms for the optimization of elution flowrate 91
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4.11 The relationship between recovery percentage and loading
capacity 93
4.12 HIC chromatograms for the optimization of rhTf loading
capacity 94
4.13 SDS-PAGE characterizing the elution profile of rhTf 95
4.14 Binding capacity of two anion exchange matrix with Tris and
phosphate buffer used as equilibration buffer 96
4.15 Binding capacity of Q-Sepharose with equilibration buffer of
different pH 97
4.16 Binding capacity of Q-Sepharose with different concentration
of equilibration buffer 97
4.17 Anion exchange chromatograms for the optimization of
selectivity 99
4.18 SDS-PAGE characterizing the elution profile of rhTf 100
4.19 HIC chromatogram characterizing the separation and elution
profile of sample 102
4.20 SDS-PAGE characterizing the separated protein from phenyl
sepharose 6 fast flow column 102
4.21 Anion exchange chromatogram characterizing the separation
and elution profile of sample of after HIC and after dialysis 103
4.22 SDS-PAGE characterizing the separated protein from
Q-Sepharose column 103
4.23 SDS-PAGE characterizing the sample pooled from each
purification step 104
4.24 Chromatogram shows HPLC separation of PMP-labeled
transferrin 106
4.25 Standard calibration graph of monosaccharides 107
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LIST OF SYMBOLS/ ABBREVIATIONS
% Percentage
α Alpha
β Beta
µm Micro meter
°C Degree Celsius
µg Micro gram
µg/ml Micro gram per milliliter
µl Microliter.
µm Micrometer
µmol/ml Micro mol per milliliter
AAGR Average annual growth rate
Ablank Absorbance for blank
AcMNPV Autographa californica multiple nuclear polyhedrosis virus
ACN Acetonitrile
AcNPV Autographa californica nuclear polyhedrolysis
Asample Absorbance for sample
Asn-X-Ser Asparagine-X-Serine
Asn-X-Thr Asparagine-X-Threonine
Astandard Absorbance for standard
ATCC American Tissue Culture Collection
BEVS Baculovirus expression vector system
BHK Baby hamster kidney cells
Bm Bombyx mori
BmNPV Bombyx mori nuclear polyhedrosis virus.
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BV Budded virus
BmNPV Bombyx mori nuclear polyhedrosis virus.
BV Budded virus
BVs Budded viruses
cDNA Complementary deoxyribonucleic acid
cells/ml Cells per milliliter
CHO Chinese Hamster Ovary
CM Carboxymethyl
cm/hr Centimeter per hour
cm2 Centimeter square
CMP-NeuAc Cytidine-5’-monophospho N-acetylneuraminic acid
Cu1+
Cuprous ion
CuSO4•5H2O Copper (II) sulfate pentahydrate
CV Column Volume
DEAE Diethylaminoethyl
DMSO Dimethyl sulphoxide
DNA Deoxyribonucleic Acid
DO Dissolved oxygen
DPA Dipicolylamine
e- Electron
E.coli Escherichia coli
ELISA Enzyme linked immunorsorbent assay
ER Endoplasmic recticulum
FBS Fetal bovine serum
FDA Food and Drugs Administration
Fe3+
Ferric ion
Fuc Fucose
g Gravitational
g/l Gram per liter
Gal Galactose
GalNAc N-Acetylgalactosamine
GDP-mannose Guanosine diphoshate mannose
GlcN Glucosamine
GlcNAc N-Acetylglucosamine
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GLDH Glutamate dehydrogenase
GLP-1 Glucagons-like peptide 1
GLP-1-R Glucagons-like peptide 1-receptor
GMP Good manufacturing practice
gp Glycoprotein
GV Granuloviruses (GV)
H+ Hydrogen cation
H2O2 Hydrogen peroxide
H3PO4 Phosphoric acid
HIC Hydrophobic interaction chromatography
His6 Hexahistidine
HPLC High performance Liquid Chromatografi
HRP Horseradish peroxidase
Hrs Hours
hTf Human transferrin
IEX Ion exchange chromatography
IgG Immunoglobulin G
IMAC Metal affinity chromatography
k constant
Kb/kbp Kilo base pair
kDa Kilo Dalton
M Molar
Man Mannose
Man3–1GlcNAc2 3(Mannose)-2(N-Acetyl Glucosamine)
Man3GlcNAc2 3(Mannose)-2(N-Acetylglucosamine)
Man8–GlcNAc2 8(Mannose)-2(N-Acetylglucosamine)
Man9GlcNAc2 9(Mannose)-2(N-Acetylglucosamine)
MeOH Methanol
mg Milligram
mg/ml Milligram per milliliter
min Minutes
ml/min Milliliter per minutes
mmol/L milli mol per liter
MOI Low multiplicity of infection
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MPa Mega Pascal
MW Molecular weight
MWCO Molecular Weight Cut Off
N Normal
N.D Not defined
NaCl Sodium Chloride
NADP+/NADPH Nicotinamide adenine dinucleotide phosphate
Na3C6H5O7 Sodium citrate
NaOH Sodium hydroxide
ng/ml Nanogram per milliliter
NH3 Ammonia
(NH4)2SO4 Ammonium Sulphate
Ni2+
Nickel ion
nm Nano meter
NPV Nucleopolyhedoviruses
O2 Oxygen
OB Occlusion bodies
ODS Octadecyl silica
ODV Occlusion derived virus
OV Occluded virus
p10 Phage-encoded protein-10
PBS Phosphate buffered saline
pfu/ml Plug performing unit per milliliter
pH Potential hydrogen
pI Isoelectric point
PIBs Polyhedral inclusion bodies
pmol Pico mol
PMP 1-Phenyl-3-Methyl-5-Pyrazolone
QAE Quaternary Aminoethyl
Q-sepharose Quaternary ammonium
rhTf Recombinant human transferrin
RP-HPLC Reversed phase HPLC
rpm Rotation per minutes
RT Retention time
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S Methyl sulphonate
S. cerevisiae Saccharomyces cerevisiae
SDS Sodium dodecyl sulfate
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SFM Serum Free Medium
SP Sulphopropyl
T.ni Trichoplusia ni
TBS Tris buffered saline
TCID50 50 % Tissue Culture Infectious Dose
TCID50/ml 50 % Tissue Culture Infectious Dose per milliliter
TEMED N,N,N',N'-tetramethylethylenediamine
TFA Trifluoroacetic acid
TM Trademark
TMB 3,3’,5,5’-tetramethylbenzidene
TN5B1-4 High 5
TOI Time of Infection
Tris-HCl Tromethamine and Hydrochloric Acid
UDP Uridine-5’-diphophate
UDP-Gal Uridine-diphosphate galactose
UDP-Glc Uridine-diphosphate glucose
UDP-GlcNAc Uridine-diphophate N-acetylglucosamine
V Volts
W.R Working reagent
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LIST OF APPENDICES
APPENDIX NO. TITLE PAGE
A-1 Stock Solution for SDS-PAGE 134
A-2 Working Solution for SDS-PAGE 135
A-3 Separating and Stacking Gel Preparation 136
B Coomassie Blue Staining 137
C Preparation of Optimized Medium 138
D Example of TCID50 Calculation (spreadsheet) 139
E Working Solution for ELISA 141
F Working Solution for Western Blot 143
G Mobile Phase for Purification 144
H Glycan Analysis 145
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CHAPTER 1
INTRODUCTION
1.1 Preface
The biopharmaceutical industry has experienced a significant transformation
based on the development of recombinant DNA and hybridoma technologies in the
1970s. The industry has moved beyond simple replication of human proteins (such
as insulin or growth hormones) and played a key role in the development of large-
molecule drugs such as any protein, virus, therapeutic serum, vaccine, and blood
component. These genetically engineered therapeutic drugs are targeting some of the
major illnesses such as cancer, cardiovascular, and infectious diseases and they have
the full potential to tackle a whole array of new diseases effectively and safely.
By mid 2003, 148 biopharmaceuticals proteins were approved in the United
States and Europe compared to 84 in 2000 (Birch and Onakunle, 2005). The total
global market for protein drugs was $47.4 billion in 2006 and the market is presumed
to reach $55.7 billion by the end of 2011 with an average annual growth rate
(AAGR) of 3.3% (Figure 1.1). It is expected that current cell culture facilities are
unlikely to meet expected demand. The imbalance of supply-demand is
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expected to get worse in the future, as more biotech therapeutics proteins are
approved. 20–50% of potential therapeutics could be delayed due to the lack of
manufacturing capacity (Fernandez et al., 2002). Hence, the ability in expanding the
existing capacity and producing a larger variety of products are crucial in order to
meet future demand. Drug companies and biotech firms are considering alternative
manufacturing platforms, besides increasing fermentation capacity (Table 1.1)
(Elbehri, 2005).
Figure 1.1: Worldwide sales forecast for protein drugs, 2006 and 2011 (Talukder,
2007).
Generally, recombinant therapeutic protein can be generated and produced in
various prokaryotic and eukaryotic expression systems. Until the early 1990s, the
majority of recombinant proteins were expressed in either microbial or mammalian
cell culture systems. The first approved recombinant therapeutic glycoproteins,
insulin is produced from Escherichia coli. Today, the manufacturing of
biotechnology products relies heavily on the use of mammalian cells, chiefly on
Chinese Hamster Ovary (CHO) cells. The well-known drugs Avonex (interferon
beta 1-a, Biogen, Inc) and EPOGEN/EPREX (epoetin alfa, Amgen Inc/ Ortho
Biotech) are produced in CHO. Insect, transgenic plant, transgenic animal and yeast
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cells are also attractive as hosts for the production of recombinant proteins, as they
represent potentially inexpensive and versatile expression systems. Optimal
expression system can be varied, based on different critical parameters of the protein
of interest. Selecting an appropriate expression system for the protein of interest will
affect factors such as time to market, cost of goods, product characteristics,
regulatory hurdles, and intellectual property (Figure 1.2).
Table 1.1: Comparison of pharmaceutical expression system (Elbehri, 2005).
Expression System Advantages Disadvantages Applications
Cost per gram
Bacteria
Established regulatory track; well-understood genetics; cheap and easy to grow
Proteins not usually secreted; contain endotoxins; no posttranslational modifications
Insulin (E. coli; Eli Lilly); growth hormone (Genentech); growth factor; interferon
N.R
Yeast
Recognized as “safe;” long history of use; fast; inexpensive; posttranslational modifications
Overglycosylation can ruin bioactivity; safety; potency; clearance; contains immunogens/antigens
Beer fermentation; recombinant vaccines; hepatitis B viral vaccine; human insulin
$50-100
Insect cells
Posttranslational modifications; properly folded proteins; fairly high expression levels
Minimal regulatory track; slow growth; expensive media; baculovirus infection (extra step); mammalian virus can infect cells
Relatively new medium; Novavax produces virus-like particles
N.R
Mammalian cells
Usually fold proteins properly; correct posttranslation modifications; good regulatory track record; only choice for largest proteins
Expensive media; slow growth; may contain allergens/ contaminants; complicated purification
Tissue plasminogen activator; factor VIII (glycoprotein); monoclonal antibodies (Hercepin)
$500–5,000
Transgenic animals
Complex protein processing; very high expression levels; easy scale up; low-cost production
Little regulatory experience; potential for viral contamination; long time scales; isolation/GMPs on the farm
Lipase (sheep, rabbits; PPL Therapeutics); growth hormone (goats; Genzyme); factor VIII (cattle)
$20–50
Transgenic plants
Shorter development cycles; easy seed storage/scaling; good expression levels; no plant viruses known to infect humans
Potential for new contaminants (soil fungi, bacteria, pesticides); posttranslational modifications; contains possible allergens
Cholera vaccine (tobacco; Chlorogen, Inc.); gastric lipase (corn; Meristem); hepatitis B (potatoes; Boyce Thompson)
$10–20
N.R- Not Reported
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4
The baculovirus expression vector system (BEVS) has a number of
significant advantages over other methods of recombinant protein production. It is
best known as providing quick access to biologically active proteins and used as a
research tool (Cox, 2004). The major advantages of BEVS over bacterial and
mammalian expression system is the very high expression of recombinant proteins
which in many cases are antigenically, immunogenically and functionally similar to
their native counterparts (Goosen, 1993). Lack of adventitious viral agents that
could replicate in mammalian cells (John Morrow, 2007), make BEVS a powerful
manufacturing platform for health care solutions to pandemic, biodefense, and
emergency scenarios (Cox, 2004). However, BEVS also has its limitation in
producing authentic mammalian proteins and glycoproteins. An absence of complex
sugars in BEVS-produced proteins may result in poor pharmacological activity in
vivo due to the rapid clearance from the circulatory system of glycoproteins with
non-human glycans (Betenbaugh et al., 2004)
Figure 1.2: Strength and weaknesses of various expression systems (Cox, 2004).
The deficiency of BEVS in producing mammalian like-glycoproteins of
potential therapeutic is a hot topic among researchers in this field. BEVS had been
reported to produce sialylated complex type N-glycan through the modification of its
metabolic engineering pathway (Betenbaugh et al., 2004; Viswanathan et al., 2005;
Page 26
5
Yun et al., 2005). Protein Sciences Corporation (PSC) had developed technology for
large-scale (600 L) production of proteins in insect cells using the BEVS (Cox,
2004). Although currently there are no FDA-approved therapeutic proteins
expressed using BEVS, a number of products are in advanced clinical trials and
several are about to get acceptance. Among these, three vaccines that are close to
market are Provenge™, a prostate cancer immunotherapy from Dendreon
(www.dendreon.com); Ceravix™, a papilloma virus vaccine from GlaxoSmithKline
(www.gsk.com); and FluBIOk™ from Protein Sciences, a non-egg based flu vaccine
(John Marrow, 2007).
BEVS have tremendous potential to become the next therapeutic
manufacturing system. In this study, recombinant human transferrin was used as a
model protein. Transferrin was chosen because of the simplicity of its structure and
its recent important role in protein engineering. Non-glycosylated transferrin had
been used as a scaffold to extend the half life of peptide and proteins. Various
chromatographic methods for purification of transferrin have been reported. Among
these reports, Ali et al. (1996) and Ailor et al. (2000) had purified rhTf from sf9 and
Tn cells using phenyl sepharose and Q-Sepharose. In this study, hydrophobic
interaction chromatography utilizing phenyl sepharose was used as the capture step
and IEX chromatography utilizing Q-sepharose was used for further purification of
rhTf. To obtain pure rtTf, optimization of both chromatographic techniques had
been carried out. Basic characterization of the carbohydrate content of the pure rhTf
had also been carried out to get a better understanding of the glycan.
Page 27
114
Superoxide and Hydrogen Peroxide. The Journal of Biological Chemistry.
259, 13391-13394.
Bauer, H. C., Parent, J. B. and Olden, K. (1985). Role of Carbohydrate in
Glycoprotein Secretion by Human Hepatoma Cells. Biochemical and
Biophysical Research Communications. 128, 368-375.
Beare, S. and Steward, W.P. (1996). Plasma Free Iron and Chemotherapy Toxicity.
The Lancet. 347, 342–343.
Bedard, C., Tom, R. and Kamen, A. (1993). Growth, Nutrient Consumption and End-
Product ccumulation in Sf9 and BTI-EAA Insect Cell Cultures: Insights into
Growth Limitation and Metabolism. Biotechnology Progress. 9, 615-624.
Belew, M., Yafang, M., Bin, L., Berglöf, J. and Janson, J-C. (1991). Purification of
Recombinant Hepatitis B Surface Antigen Produced by Transformed Chinese
Hamster Ovary (CHO) Cell Line Grown in Culture. Bioseparation. 1, 397–
408.
Beljelarskaya, S.N. (2002). A Baculovirus Expression System for Insect Cells.
Molecular Biology. 36(3), 281–292.
Betenbaugh, M.J, Tomiya, N., Narang, S., Hsu, J.T.A. and Lee, Y.C. (2004).
Biosynthesis of Human-Type N-Glycans in Heterologous Systems. Current
Opinion in Structural Biology. 14,601–606.
Birch, J.R. and Onakunle, Y. (2005). Biopharmaceutical Proteins: Opportunities and
Challenges. In: Mark Smales, C. and James, D.C. Therapeutic Proteins:
Methods and Protocols. New Jersey: Humana Press.
Bonnerjera, J., Oh, S., Hoare, M. and Dunhill, P. (1986). The Right Step at The Right
Time. Bio/Technology. 4, 954-958.
Page 28
115
Bradley, S.J., Gosriwatana, I., Srichairantankool, S., Hider, R.C. and Porter, J.B.
(1997). Non-transferrin-bound Iron Induced by Myeloablative Chemotherapy.
British Journal of Haematology. 99, 337–343.
Breitbach, K., Jarvis, D.L. (2001). Improved Glycosylation of A Foreign Protein by
Tn-5B1-4 Cells Engineered to Express Mammalian Glycosyltransferases.
Biotechnology And Bioengineering. 74(3), 230-239.
Bullen, J.J., Griffiths, E. (1999). Iron and Infection, Molecular, Physiological and
Clinical Aspects. (2nd Edition). Chichester: John Wiley and Sons.
Burgess, S. (1977). Molecular Weights of Lepidopteran Baculovirus DNAs:
Derivation by Electron Microscopy. The Journal of General Virology. 37,
501-510.
Carson, D.D. (1992) Proteoglycans in Development. In: Fukuda, M. (Ed.). Cell
Surface Carbohydrates and Cell Development. (pp. 257-274). London: CRC
Press.
Castro, P.M.L., Ison, A.P., Hayte, P.M. and Bull, A.T. (1995). The
Macroheterogeneity of Recombinant Human Interferon-γ Produced by
Chinese Hamster Ovary Cells is Affected by The Protein and Lipid Content
of The Culture Medium. Biotechnology and Applied Biochemistry. 21, 87-
100.
Chauhan, A., Chauhan, V., Brown, W.T. and Cohen, I. (2004). Oxidative Stress in
Autism: Increased Lipid Peroxidation and Reduced Serum Levels of
Ceruloplasmin and Transferrin – The Antioxidant Proteins. Life Sciences. 75,
2539–2549.
Chiou, T.W., Hsieh, Y.C. and Ho, C.S. (2000). High Density Culture of Insect Cells
using Rational Medium Design and Feeding Strategy. Bioprocess
Engineering. 22, 483-491.
Page 29
116
Choi, O., Tomiya, N., Kim, J.H., Slavicek, J.M., Betenbaugh, M.J. and Lee, Y.C.
(2003). N-Glycan Structures of Human Transferrin Produced by Lymantria
dispar (Gypsy moth) Cells using The LdMNPV Expression System.
Glycobiology. 13(7), 539-548.
Choudhury, D., Thakurta, P.G., Dasgupta, R., Sen, U., Biswas, S., Chakrabarti, C.
and Dattagupta, J.K. (2002). Purification and Preliminary X-ray Studies on
Hen Serotransferrin in Apo- and Holo-Forms. Biochemical and Biophysical
Research Communications. 295,125–128.
Chu, L. and Robinson, D.K. (2001). Industrial Choices for Protein Production by
Large Scale Cell Culture. Current Opinion in Biotechnology. 12: 180-187
Comings, D.E., Miguel, A.G. and Lesser, H.H. (1979). Nuclear proteins. VI.
Fractionation of Chromosomal Non-Histone Proteins using Hydrophobic
Chromatography. Biochimica et Biophysica. Acta. 563, 253–260.
Cox, M.M.J (2004). Chapter 3: Commercial Production in Insect Cells-One
Company’s Perspective. Bioprocess International. 2(2), 34-38.
Crowther, J.R. (1995). Elisa: Theory and Practice. Totowa, New Jersey: Human
Press.
Cumming, D.A. (1992). Physiological Relevance of Protein Glycosylation.
Development in Biological. Standardization. 76, 83-94
Cummings, R.D., Merkle, R.K. and Stults, N.L. (1989). Separation and analysis of
glycoprotein oligosaccharides. Methods in Cell Biology. 32, 141-183.
Davies, A.H. (1994). Current Methods for Manipulating Baculoviruses.
Bio/Technology. 12, 47–50.
Page 30
114
Superoxide and Hydrogen Peroxide. The Journal of Biological Chemistry.
259, 13391-13394.
Bauer, H. C., Parent, J. B. and Olden, K. (1985). Role of Carbohydrate in
Glycoprotein Secretion by Human Hepatoma Cells. Biochemical and
Biophysical Research Communications. 128, 368-375.
Beare, S. and Steward, W.P. (1996). Plasma Free Iron and Chemotherapy Toxicity.
The Lancet. 347, 342–343.
Bedard, C., Tom, R. and Kamen, A. (1993). Growth, Nutrient Consumption and End-
Product ccumulation in Sf9 and BTI-EAA Insect Cell Cultures: Insights into
Growth Limitation and Metabolism. Biotechnology Progress. 9, 615-624.
Belew, M., Yafang, M., Bin, L., Berglöf, J. and Janson, J-C. (1991). Purification of
Recombinant Hepatitis B Surface Antigen Produced by Transformed Chinese
Hamster Ovary (CHO) Cell Line Grown in Culture. Bioseparation. 1, 397–
408.
Beljelarskaya, S.N. (2002). A Baculovirus Expression System for Insect Cells.
Molecular Biology. 36(3), 281–292.
Betenbaugh, M.J, Tomiya, N., Narang, S., Hsu, J.T.A. and Lee, Y.C. (2004).
Biosynthesis of Human-Type N-Glycans in Heterologous Systems. Current
Opinion in Structural Biology. 14,601–606.
Birch, J.R. and Onakunle, Y. (2005). Biopharmaceutical Proteins: Opportunities and
Challenges. In: Mark Smales, C. and James, D.C. Therapeutic Proteins:
Methods and Protocols. New Jersey: Humana Press.
Bonnerjera, J., Oh, S., Hoare, M. and Dunhill, P. (1986). The Right Step at The Right
Time. Bio/Technology. 4, 954-958.
Page 31
115
Bradley, S.J., Gosriwatana, I., Srichairantankool, S., Hider, R.C. and Porter, J.B.
(1997). Non-transferrin-bound Iron Induced by Myeloablative Chemotherapy.
British Journal of Haematology. 99, 337–343.
Breitbach, K., Jarvis, D.L. (2001). Improved Glycosylation of A Foreign Protein by
Tn-5B1-4 Cells Engineered to Express Mammalian Glycosyltransferases.
Biotechnology And Bioengineering. 74(3), 230-239.
Bullen, J.J., Griffiths, E. (1999). Iron and Infection, Molecular, Physiological and
Clinical Aspects. (2nd Edition). Chichester: John Wiley and Sons.
Burgess, S. (1977). Molecular Weights of Lepidopteran Baculovirus DNAs:
Derivation by Electron Microscopy. The Journal of General Virology. 37,
501-510.
Carson, D.D. (1992) Proteoglycans in Development. In: Fukuda, M. (Ed.). Cell
Surface Carbohydrates and Cell Development. (pp. 257-274). London: CRC
Press.
Castro, P.M.L., Ison, A.P., Hayte, P.M. and Bull, A.T. (1995). The
Macroheterogeneity of Recombinant Human Interferon-γ Produced by
Chinese Hamster Ovary Cells is Affected by The Protein and Lipid Content
of The Culture Medium. Biotechnology and Applied Biochemistry. 21, 87-
100.
Chauhan, A., Chauhan, V., Brown, W.T. and Cohen, I. (2004). Oxidative Stress in
Autism: Increased Lipid Peroxidation and Reduced Serum Levels of
Ceruloplasmin and Transferrin – The Antioxidant Proteins. Life Sciences. 75,
2539–2549.
Chiou, T.W., Hsieh, Y.C. and Ho, C.S. (2000). High Density Culture of Insect Cells
using Rational Medium Design and Feeding Strategy. Bioprocess
Engineering. 22, 483-491.
Page 32
116
Choi, O., Tomiya, N., Kim, J.H., Slavicek, J.M., Betenbaugh, M.J. and Lee, Y.C.
(2003). N-Glycan Structures of Human Transferrin Produced by Lymantria
dispar (Gypsy moth) Cells using The LdMNPV Expression System.
Glycobiology. 13(7), 539-548.
Choudhury, D., Thakurta, P.G., Dasgupta, R., Sen, U., Biswas, S., Chakrabarti, C.
and Dattagupta, J.K. (2002). Purification and Preliminary X-ray Studies on
Hen Serotransferrin in Apo- and Holo-Forms. Biochemical and Biophysical
Research Communications. 295,125–128.
Chu, L. and Robinson, D.K. (2001). Industrial Choices for Protein Production by
Large Scale Cell Culture. Current Opinion in Biotechnology. 12: 180-187
Comings, D.E., Miguel, A.G. and Lesser, H.H. (1979). Nuclear proteins. VI.
Fractionation of Chromosomal Non-Histone Proteins using Hydrophobic
Chromatography. Biochimica et Biophysica. Acta. 563, 253–260.
Cox, M.M.J (2004). Chapter 3: Commercial Production in Insect Cells-One
Company’s Perspective. Bioprocess International. 2(2), 34-38.
Crowther, J.R. (1995). Elisa: Theory and Practice. Totowa, New Jersey: Human
Press.
Cumming, D.A. (1992). Physiological Relevance of Protein Glycosylation.
Development in Biological. Standardization. 76, 83-94
Cummings, R.D., Merkle, R.K. and Stults, N.L. (1989). Separation and analysis of
glycoprotein oligosaccharides. Methods in Cell Biology. 32, 141-183.
Davies, A.H. (1994). Current Methods for Manipulating Baculoviruses.
Bio/Technology. 12, 47–50.