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 :

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

iii

To my beloved grandparents, parents and brothers

iv

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.

v

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.

vi

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.

vii

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

viii

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

ix

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

x

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

xi

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

xii

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

xiii

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

xiv

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

xv

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

xvi

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.

xvii

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

xviii

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

xix

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

xx

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

xxi

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

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

2

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

3

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

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;

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.

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.

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.

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.

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.

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.

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.