UNIVERSITI PUTRA MALAYSIA

FADZLIE WONG FAIZAL WONG

FK 2010 73

SIMPLE SURFACE PLASMON RESONANCE BIO-SENSOR FOR THE QUANTIFICATION OF RECOMBINANT HUMAN EPIDERMAL GROWTH

FACTOR (rhEGF) IN EXPANDED BED RECOVERY

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SIMPLE SURFACE PLASMON RESONANCE BIO-SENSOR FOR THE

QUANTIFICATION OF RECOMBINANT HUMAN EPIDERMAL GROWTH

FACTOR (rhEGF) IN EXPANDED BED RECOVERY

By

FADZLIE WONG FAIZAL WONG

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

In Fulfilment of the Requirements for the Degree of Master of Science

September 2010

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With the name of ALLAH The Almighty,

For the sake of learning and knowledge……..

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of

the requirement for the degree of Master of Science

SIMPLE SURFACE PLASMON RESONANCE BIO-SENSOR FOR THE

QUANTIFICATION OF RECOMBINANT HUMAN EPIDERMAL GROWTH

FACTOR (rhEGF) IN EXPANDED BED RECOVERY

By

FADZLIE WONG BIN FAIZAL WONG

September 2010

Chairman: Associate Professor Ling Tau Chuan, PhD

Institute: Faculty of Engineering

The conventional quantification assays used to monitor the performance of protein

downstream processing are typically not user friendly as it is laborious, time

consuming and costly. At the same time, the emergence of multi-modal adsorbent,

Streamline Direct HST (1.8 g/cm3) to handle high ionic strength feedstock can

potentially improve the recovery of protein in expanded bed adsorption (EBA).

Hence, the objectives of the study are to develop a reliable quantification assay for

recombinant human epidermal growth factor (rhEGF) by Surface Plasmon Resonance

and to examine its application for the monitoring of rhEGF recovery by HST

adsorbent in EBA. It was developed based on the BIAcore 3000 instrument with anti-

rhEGF antibody immobilized on the CM5 chip. The performances of the assay were:

assay linearity (25 to 250 ng/mL), accuracy (within 10% recovery of target), precision

(below 3.4% CV), intra- and inter-assay precision (less than 20% CV) and

quantification limit (8.0 ng/mL). In EBA study, the optimum binding condition of

rhEGF onto Streamline HST was at pH 4 in acetate buffer without the NaCl or at high

(1.8 M) NaCl concentration as determined from the batch binding experiment. The

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electrostatic interaction, hydrophobic interaction and the hydrogen bonds are

responsible for this. As for the elution, potassium buffer with pH 12 was used

exploiting the effect of electrostatic repulsion. The effect of cells (Escherichia coli

and Pastoris pastoris) on the performance of HST adsorbent in capturing rhEGF was

also being examined by fitting the adsorption into the Langmuir isotherm. The

thermodynamic parameters: maximum binding capacity (qm) and equilibrium

dissociation constant (Kd) are little affected as the concentration of these biomass

increased from 0 to 4.73 % w/v. The result showed that the adsorption level was not a

strong function of the biomass concentration. Next, the feasibility of using the HST

adsorbent to capture rhEGF in EBA was finally tested using the single component

sample (only rhEGF) with the presence of biomass (4.73 % w/v E. coli or P.

pastoris). Fastline 10 column (Upfront, Denmark) was used to handle 20 mL of

sample volume with adsorbent settled bed height of 2 cm (1.6 mL). The results

demonstrated that a consistent yield can be obtained even with the presence of cells

(97.38% for control, 96.94% for P. pastoris and 96.43% for E. coli) and a stable bed

can be achieved, suggesting that no interference of adsorption by biomass occurred. In

conclusion, the major contributions of the study are the development of reliable SPR

quantification assay and the EBA process template that is useful for rhEGF

purification from real feedstock.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

BIO-PENDERIA ALUNAN PLASMON PERMUKAAN MUDAH BAGI

PENJUMLAHAN REKOMBINAN FAKTOR PERTUMBUHAN

EPIDERMAL MANUSIA (rhEGF) DALAM PEROLEHAN LAPISAN

TERKEMBANG

By

FADZLIE WONG BIN FAIZAL WONG

September 2010

Pengerusi: Profesor Madya Ling Tau Chuan, PhD

Institut: Fakulti Kejuruteraan

Cerakin pengukuran konvensional yang digunakan untuk memantau prestasi

pemprosesan hiliran protein biasanya tidak mesra pengguna disebabkan ianya

memerihkan, memakan masa dan mahal. Di samping itu, kemunculan penjerap multi-

modal, Streamline Direct HST (1.8 g/cm3) untuk menangani stok suapan berkekuatan

ionik tinggi berpotensi untuk memajukan proses pemulihan protin di dalam expanded

bed adsorption (EBA). Maka, objektif kajian ini adalah untuk membangunkan satu

cerakin pengukuran bagi rekombinan faktor pertumbuhan epidermal (rhEGF) yang

boleh dipercayai dan untuk diaplikasikan dalam pemantauan proses pemulihan rhEGF

dengan penjerap HST di dalam EBA. Ia adalah berdasarkan instrumen BIAcore 3000

dengan antibodi anti-rhEGF yang disekatgerakkan pada cip CM5. Prestasi cerakin

tersebut adalah: julat linear (25 to 250 ng/mL), ketepatan (dalam 10% perolehan

target), kejituan (bawah 3.4% CV), kejituan intra- dan inter-assay (kurang daripada

20% CV) dan had penentuan (8.0 ng/mL). Dalam kajian EBA, keadaan pengikatan

yang optimum untuk rhEGF pada penjerap HST adalah pada pH 4 dalam penimbal

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asetat dengan tiada NaCl atau dengan kepekatan NaCl yang tinggi (1.8 M) seperti

yang telah ditentukan oleh eksperimen pengikatan kelompok. Interaksi elektrostatik,

hidrofobik dan ikatan hidrogen bertanggungjawab untuk ini. Bagi proses elusi,

penimbal potassium dengan pH 12 digunakan dengan mengekspoitasi kesan

penolakan elektrostatik. Kesan kehadiran sel (Escherichia coli and Pichia pastoris) ke

atas prestasi penjerap HST dalam mengikat rhEGF juga diperiksa dengan

menyesuaikan proses penjerapan dengan Langmuir isotherm. Parameter-parameter

termodinamik: kapasiti pengikatan maksimum (qm) dan pemalar penceraian

keseimbangan (Kd) terjejas sedikit apabila kepekatan bagi sel-sel tersebut

ditingkatkan daripada 0 hingga 4.73 % w/v. Keputusan tersebut menunjukkan bahawa

tahap penjerapan tidak bergantung kepada kepekatan sel. Kemudian, kebolehan untuk

menggunakan penjerap HST dalam mengikat rhEGF di dalam EBA akhirnya diuji

dengan menggunakan sampel satu komponen (hanya rhEGF) dengan kehadiran sel

(4.73 % w/v E. coli or P. pastoris). Turus Fastline 10 (Upfront, Denmark) digunakan

untuk menangani 20 mL isipadu sampel dengan 2 cm (1.6 mL) ketinggian lapisan

termendak. Keputusan tersebut menunjukkan bahawa hasil yang konsisten boleh

diperolehi walaupun dengan kehadiran sel (97.38% bagi kawalan, 96.94% bagi P.

pastoris dan 96.43% bagi E. coli) dan lapisan yang stabil boleh dicapai, menunjukkan

bahawa tiada gangguan penjerapan oleh biomas yang berlaku. Kesimpulannya,

sumbangan utama kajian ini adalah pembangunan cerakin pengukuran SPR yang

boleh dipercayai dan templat proses EBA yang berguna untuk penulenan rhEGF

daripada stok suapan sebenar.

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ACKNOWLEDGEMENTS

Grateful to Allah Subhanahuwataala, who mercifully favored me to accomplish this

work. With his bless, thanks to Him for this valuable experience.

I would like to thank my supervisor, Assoc. Prof. Dr. Ling Tau Chuan for his

guidance and attention given to me during the study. The same also goes to my other

supervisory committee members: Prof. Dr. Arbakariya Ariff, Assoc. Prof. Dr. Zarida

Hambali and Dr. Siti Mazlina. It was through their critical comments and ideas that

significantly helping me to complete the study.

Furthermore, I would also like to thank Ms. Arbaah Salleh, Mr. Ismail Baharom, Mr.

Hanif Md. Arshad, Ms. Norhayati and Mr. Daud Yusof for their help and assistance to

get myself settled and enjoying working in the Laboratory of Immunotherapeutic and

Vaccines (LIVES).

In addition I would also like to mention names of my colleagues at the laboratory who

has creating such a harmony environment, providing helps, comments, support and

most importantly the friendship. They are Faizal, Elysha, Tam, Lo, Ira, Nik, Joo Shun,

Ani, Chien Wei, Fatemeh, Tayeb, Ramaman, Ruzila, Parisa, Morvarid, Hidayah, Zaid,

Razif, Kelvin, Dr. Hamid, Hoe Shee, Yu Kiat and Joo Ee.

Last but not least, I also would like to show my gratitude to Ministry of Higher

Education (UPM) who sponsors my study in this Master programme through the

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SLAB scheme. Then it is also deserved to highlight the contribution of Ministry of

Science, Technology and Innovation (MOSTI) for the IRPA grant that allow this

research to be carried out.

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I certify that an Examination Committee has met on 2nd

September 2010 to conduct

the final examination of Fadzlie Wong Faizal Wong on his Master of Science thesis

entitled “A simple surface plasmon resonance bio-sensor for the quantification of

recombinant human epidermal growth factor (rhegf) in expanded bed recovery” in

accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and

Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee

recommends that the student be awarded the Master of Science.

Members of the Examination Committee were as follows:

Johari Endan, PhD

Associate Professor

Faculty of Engineering

Universiti Putra Malaysia

(Chairman)

Chin Nyuk Ling, PhD

Associate Professor

Faculty of Engineering

Universiti Putra Malaysia

(Internal Examiner)

Rosfarizan Mohamad, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Internal Examiner)

Shaliza Ibrahim, PhD

Professor

Faculty of Engineering

Universiti Malaya

(External Examiner)

______________________________

SHAMSUDDIN SULAIMAN, PhD

Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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This thesis was submitted to Senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

Ling Tau Chuan, PhD

Associate Professor

Faculty of Engineering

Universiti Putra Malaysia

(Chairman)

Arbakariya Ariff, PhD

Professor

Faculty of Biotechnology and Biomolecular Sciences

University Putra Malaysia

(Member)

Zarida Hambali, PhD

Associate Professor

Ministry of Higher Education

(Member)

Siti Mazlina Mustapa Kamal, PhD

Lecturer

Faculty of Engineering

Universiti Putra Malaysia

(Member)

_______________________________

HASANAH MOHD GHAZALI, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations which

have been duly acknowledged. I also declare that it has not been previously, and is not

currently, submitted for any other degree at Universiti Putra Malaysia or at any other

institution.

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FADZLIE WONG FAIZAL WONG

Date: 2 September 2010

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TABLE OF CONTENTS

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW

2.1 Epidermal growth factor characterization 6

2.2 The application of human epidermal growth factor 8

2.3 Periplasmic production of recombinant proteins in E. coli 8

2.4 Fusion protein technology and its advantages 9

2.5 Drawbacks in fusion protein technology 10

2.6 FLAG-tag application 11

2.7 Anti-FLAG antibodies 13

2.8 The fundamental of affinity chromatography 15

2.9 Production and purification of human epidermal growth factor 18

2.10 Advances in primary purification step 28

2.10.1 Immobilized metal affinity chromatography (IMAC) 29

2.10.2 Dye-ligand chromatography 32

2.10.3 Separation of biomolecules by monoliths 34

2.10.4 Molecular imprinting 35

2.10.5 Aqueous two-phase extraction (ATPE) 37

2.10.6 Expanded bed adsorption chromatography (EBA) 39

3 DETECTION AND QUANTIFICATION ASSAY FOR

RECOMBINANT HUMAN EPIDERMAL GROWTH

FACTOR (rhEGF) IN ESCHERICHIA COLI CRUDE

EXTRACT BY A SURFACE PLASMON RESONANCE

BIOSENSOR

3.1 Introduction 42

3.2 Materials and Methods

3.2.1 Materials 44

3.2.2 Preparation of sensor surfaces 44

3.2.3 Standard curve construction and sample injection 45

3.2.4 Assay performance evaluation 45

3.3 Results and Discussion

3.3.1 Preparation of sensor surfaces 47

3.3.2 Regeneration of the anti-EGF surfaces 51

3.3.3 Standard curve construction 52

3.4 Conclusion 60

Page

DEDICATION ii

ABTRACT iii

ABSTRAK v

ACKNOWLEDGEMENTS vii

APPROVAL ix

DECLARATION xi

LIST OF TABLES xiv

LIST OF FIGURES xv

LIST OF ABBREVIATIONS xvii

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4 EXPANDED BED ADSORPTION CHROMATOGRAPHY

AS A TOOL FOR rhEGF PURIFICATION

4.1 Introduction 61

4.2 Materials and Methods

4.2.1 Microorganism cultivation 64

4.2.2 Adsorbent 65

4.2.3 Study of binding condition for rhEGF 65

4.2.4 Analyses 69

4.3 Results and Discussion

4.3.1 Study of binding condition for rhEGF 71

4.3.2 Expanded bed adsorption chromatography 77

4.4 Conclusion 82

5 SUMMARY, GENERAL CONCLUSION AND

RECOMMENDATIONS FOR FUTURE RESEARCH

5.1 General Discussion

5.2 Conclusion

5.3 Recommendations for Future Research

85

86

90

REFERENCES 94

APPENDIXES 112

BIODATA OF STUDENT 122

LIST OF PUBLICATIONS 123