purification of epidermal growth factorpsasir.upm.edu.my/id/eprint/41137/1/fk 2010 73r.pdf ·...
TRANSCRIPT
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
© COPYRIG
HT UPM
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
© COPYRIG
HT UPM
ii
With the name of ALLAH The Almighty,
For the sake of learning and knowledge……..
© COPYRIG
HT UPM
iii
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
© COPYRIG
HT UPM
iv
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.
© COPYRIG
HT UPM
v
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
© COPYRIG
HT UPM
vi
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.
© COPYRIG
HT UPM
vii
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
© COPYRIG
HT UPM
viii
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.
© COPYRIG
HT UPM
ix
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:
© COPYRIG
HT UPM
x
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:
© COPYRIG
HT UPM
xi
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.
----------------------------------------------
FADZLIE WONG FAIZAL WONG
Date: 2 September 2010
© COPYRIG
HT UPM
xii
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
© COPYRIG
HT UPM
xiii
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