UNIVERSITI PUTRA MALAYSIA

IRA AMIRA BT ROSTI

IB 2013 19

RECOVERY AND PURIFICATION OF EPIDERMAL GROWTH FACTOR FROM FERMENTATION BROTH USING EXPANDED BED CHROMATOGRAPHY

RECOVERY AND PURIFICATION OF

EPIDERMAL GROWTH FACTOR FROM

FERMENTATION BROTH USING EXPANDED

BED CHROMATOGRAPHY

IRA AMIRA BT ROSTI

MASTER OF SCIENCE

UNIVERSITI PUTRA MALAYSIA

2013

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RECOVERY AND PURIFICATION OF EPIDERMAL GROWTH FACTOR

FROM FERMENTATION BROTH USING EXPANDED BED

CHROMATOGRAPHY

By

IRA AMIRA BT ROSTI

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

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

May 2013

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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

RECOVERY AND PURIFICATION OF EPIDERMAL GROWTH FACTOR

FROM FERMENTATION BROTH USING EXPANDED BED

CHROMATOGRAPHY

By

IRA AMIRA BT ROSTI

May 2013

Chairman: Prof. Arbakariya Ariff, PhD

Faculty: Institute of Bioscience

Epidermal growth factor (EGF) is a single chain polypeptide that exhibit generous

bioactivities effect on proliferation of cells in cosmetics and pharmaceutical

industries. It is well known that biopharmaceutical production cost relies very much

on purification steps. Due to therapeutic potential and high market value of EGF,

development of an efficient and cost-effective production process is required.

Integrative chromatographic technique, Expanded Bed Adsorption (EBA), may be

used to solve the separation problems related to the effect of particulates in the

recovery process. Simple and rapid quantification method of the target protein needs

to be developed for process monitoring and control during recovery and purification

processes.

Performance of EGF recovery and purification with the presence of two different cell

types, Escherichia coli (E. coli) homogenate (treated with osmotic shock) and Pichia

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pastoris (P. pastoris) intact cells, were demonstrated using EBA. Optimum

adsorption and elution experiments were performed in a batch mode. Biomass-

adsorbent interaction and stability of bed formation were evaluated using Cell

Transmission Index (CTI) and zeta potential. In order to monitor the purification

performances, surface plasmon resonance (SPR) was used for the quantification of

EGF during recovery and purification processes. An analysis method using SPR was

developed by assessing the best choice of suitable biomolecular recognition for the

quantification of EGF. Two types of EGF antibody, monoclonal (mAb) and

polyclonal (pAb), were immobilized on the surface of chip and validated for its

characteristics including specificity, range, intermediate precision and repeatability,

recovery, dilution paradox and stability. Kinetics and affinity constants of antibodies-

EGF binding were also evaluated using 1:1 Langmuir interaction model (global

fitting).

Polyclonal antibody (pAb) is more suitable to be used as a stable ligand to quantify

EGF continuously from the consideration of kinetics, binding rate and shelf life

assessment. pAb has a better affinity (KD = 7.39 x 10-9

M) than monoclonal

antibody (mAb) (KD = 9.54 x 10-10

M). From the kinetics evaluation, it was found

that pAb has a faster reaction rate during sample injection, slower dissociation rate

during buffer injection and higher level of saturation state than monoclonal antibody

(mAb). Besides, pAb has a longer shelf life and greater cycle can be run. For

purification of EGF, maximum binding of target protein can be achieved at pH 4.5

with less biomass adhesion. Recovery of EGF from E. coli homogenate gave higher

grade purification as compared to P. pastoris intact cells using the preferred elution

agents. A purification factor of 2.5 was achieved after increasing the elution

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efficiency using lower ionic strength of elute. Since 100% Cell Transmission Index

(CTI) was achieved, interaction of biomass-adsorbent could be considered as not an

obstacle. Repulsion between negative mixed mode adsorbent with negatively charge

surface of biomass at particular condition is expected due to electrostatic charge

interaction.

Results from this study have demonstrated that EBA and SPR can be used as an

attractive alternative for efficient and cost-productive procedure in the purification of

EGF from fermentation broth with less biomass adhesion. With this approach, high

overall product yield with reduced process time and cost are expected in the large

scale industrial process for direct recovery and purification of recombinant protein

from fermentation broths.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk Ijazah Master Sains

PEMULIHAN DAN PENULENAN FAKTOR PERTUMBUHAN EPIDERMAL

DARIPADA MEDIA PENAPAIAN MENGGUNAKAN SISTEM LAPISAN

TERKEMBANG

Oleh

IRA AMIRA ROSTI

Mei 2013

Pengerusi: Prof. Arbakariya Ariff, PhD

Fakulti: Institut Biosains

.

Faktor pertumbuhan epidermal adalah satu rantai polipeptida yang mempamerkan

kesan bioaktiviti pada proliferasi sel-sel dalam industri kosmetik dan

farmaseutikal. Memang diketahui bahawa kos pengeluaran biofarmaseutikal sangat

bergantung kepada langkah-langkah penulenan. Disebabkan potensi terapeutik dan

nilai pasarannya yang tinggi, pembentukan proses pengeluaran yang cekap dan kos

efektif diperlukan. Teknik kromatografi integratif, sistem lapisan terkembang boleh

digunakan untuk menyelesaikan masalah pemisahan yang berkaitan dengan kesan

zarah dalam proses pemulihan. Kaedah kuantifikasi protein yang ringkas dan cepat

perlu dibinakan untuk proses pemantauan dan kawalan semasa proses pemulihan dan

penulenan.

Prestasi pemulihan dan penulenan EGF dengan kehadiran dua jenis sel; homogenat

Escherichia coli (E. coli) (yang dirawat dengan kejutan osmosis) dan sel utuh Pichia

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pastoris (P. pastoris) telah dinyatakan dengan menggunakan EBA. Eksperimen

jerapan dan elusi optimum telah dijalankan dalam mod kelompok. Interaksi biojisim-

penjerap dan kestabilan pembentukan lapisan telah dinilai menggunakan sel

penghantaran index dan potensi zeta. Dalam usaha untuk memantau prestasi

penulenan, SPR telah digunakan untuk mengkuantitikan jumlah EGF semasa proses

pemulihan dan penulenan. Satu kaedah analisis menggunakan SPR telah

dibangunkan dengan menilai pilihan biomolekul pengecaman terbaik yang bersesuai

untuk pengkuantitian EGF. Dua jenis EGF antibodi; monoklon dan poliklon telah

dipegunkan di atas permukaan cip dan disahkan untuk ciri-ciri termasuk

kekhususan, julat, ketepatan perantaraan dan ulangan, perolehan, pencairan paradoks

dan kestabilan. Kinetik dan pemalar afiniti ikatan antibodi-EGF telah dinilai

menggunakan 1:1 Langmuir interaksi model (pemadanan global).

Antibodi poliklon (pAb) adalah lebih sesuai digunakan sebagai ligan yang stabil

untuk pengkuantitian EGF berterusan daripada pertimbangan kinetik, kadar ikatan

dan penilaian jangka hayat. pAb mempunyai afiniti yang lebih baik (KD = 7.39 x 10-9

M) daripada antibodi monoklon (mAb) (KD = 9.54 x 10-10

M). Lanjutan penilaian

pemalar kinetik menunjukkan bahawa pAb mempunyai kadar tindak balas yang lebih

cepat semasa suntikan sampel, kadar ceraian yang lebih perlahan semasa suntikan

penimbal dan peringkat ketepuan yang lebih tinggi daripada antibodi monoklon

(mAb). Selain itu, pAb mempunyai jangka hayat yang lebih panjang dan lebih

banyak kitaran boleh dijalankan. Untuk penulenan EGF, ikatan maksimum protein

sasaran boleh dicapai pada pH 4.5 dengan kurang lekatan biojisim. Pemulihan EGF

daripada homogenat E. coli memberi gred penulenan yang lebih tinggi berbanding

dengan sel utuh P. pastoris dengan menggunakan ejen elusi terbaik. Faktor

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penulenan 2.5 telah dicapai selepas kecekapan elusi ditingkatkan dengan

menggunakan elusi yang mempunyai kekuatan ionik yang lebih rendah. Interaksi

biomass-penjerap tidak menjadi halangan sementelah 100% sel penghantaran indeks

telah berjaya dicapai. Penolakan antara bahan penjerap mod campuran yang negatif

dengan permukaan caj negatif biojisim pada keadaan tertentu adalah dijangka

disebabkan oleh interaksi cas elektrostatik.

Keputusan daripada kajian ini telah menunjukkan bahawa penggunaan kaedah

pendekatan ekonomi EBA dan SPR boleh digunakan sebagai alternatif yang menarik

untuk mempunyai prosedur yang cekap dan produktif dari segi kos dalam proses

penulenan dengan kurang lekatan biojisim. Dengan pendekatan ini, keseluruhan hasil

produk dapat dipertingkatkan dengan mengambil masa yang kurang dan kos yang

lebih rendah adalah dijangka dalam pengeluaran skala perindustrian yang besar

untuk pemulihan secara langsung dan penulenan protein rekombinan daripada media

penapaian.

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ACKNOWLEDGEMENTS

Alhamdullilah, thanks to Allah for this precious experience on completing my Msc

study. This report is my thesis for a Msc degree, carried out at the Laboratory

Immunoterapeutics and Vaccines (LIVES), Universiti Putra Malaysia . This project

was supported by the IRPA grant (03-02-04-0562-SR0008/05-3) and Science fund

grant (02-01-04-SF0895) from the Ministry of Science, Technology and the

Innovation, Malaysia. (MOSTI).

I wish to express my greatest appreciation to the Chairman of my supervising

committee, Prof. Dr. Arbakariya Ariff for his guidance and supervision provided to

complete this study. I also would like to express my gratitude to the member of my

supervising committee, Prof. Dr. Ling Tau Chuan. I would like to give my grateful

acknowledgement to my senior, Dr. Ramakrishnan Nagasundara Ramanan who had

given me a lot of guidance, helpfulness, useful deliberative discussions, patience,

enthusiasm and support concerning for the completion of my Msc study. Special

thanks to all the graduate students from our department, administrative staff,

technicians and workshop men at LIVES who have an important role in creating a

place in which work is truly facilitated, as well as my friends who helped me through

this journey and with whom I had an enjoyable time in UPM.

I am specially dedicated this work to my beloved parents, Hj Rosti Mistam and Hjh

Mariam Husin, my beloved husband, Mohd Aizzuddin Md. Muhsin and my siblings

who always encouraged and supported me during different steps of my life and

education. Last but not least, thank to Allah who has made everything possible

according to His will.

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This thesis was submitted to the 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:

Arbakariya Ariff, PhD

Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Ling Tau Chuan, PhD

Professor

Faculty of Biological Science

Universiti Malaya

(Member)

BUJANG BIN KIM HUAT, 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 previously, and is not

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

other institution.

IRA AMIRA ROSTI

Date: 14 May 2013

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK v

ACKNOWLEDGEMENTS viii

APPROVAL ix

DECLARATION xi

LIST OF TABLES xv

LIST OF FIGURES xvi

LIST OF ABBREVIATIONS xix

LIST OF APPENDICES xx

CHAPTER

1 INTRODUCTION

1

2 LITERATURE REVIEW

4 2.1 General information on bioprocess separation

2.2 Integrative expanded bed adsorption 7

2.2.1 Expanded bed versus Fluidized Packed Bed operation 8

2.2.2 EBA operational procedures 12

2.3 Fouling of EBA due to biomass-adsorbent interactions 13

2.3.1 Effects of cells in feeds on the performances of EBA 14

2.3.2

Methods for the evaluation of biomass-adsorbent

interaction

16

2.3.2.1 Finite bath adsorption 18

2.3.2.2 Biomass Pulse Response 18

2.3.2.3 Resident Time Distribution (RTD) 19

2.3.2.4 Zeta Potential 19

2.4 Application of EBA for protein purification 21

2.5 Purification of hEGF 23

2.6 Epidermal growth factor (EGF) 24

2.6.1 Expression of hEGF as recombinant protein 28

2.7 Quantification methods of hEGF 29

2.8 Surface plasmon resonance (SPR) 30

2.8.1 Assay detection format 33

2.8.2 Advantages of SPR 34

2.9 Concluding remarks 37

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3 ASSESSMENT OF MOLECULAR RECOGNITION

ELEMENT FOR THE QUANTIFICATION OF HUMAN

EPIDERMAL GROWTH FACTOR USING SURFACE

PLASMON RESONANCE

3.1 Introduction 38

3.2 Materials and Methods 40

3.2.1 Materials 40

3.2.2 Surface preparation 40

3.2.3 Standard and sample preparation 41

3.2.4 Validation parameters 42

3.2.4.1 Specificity 42

3.2.4.2 Range 42

3.2.4.3 Repeatability and Precision 42

3.2.4.4 Recovery 43

3.2.4.5 Dilution Paradox 43

3.2.4.6 Robustness 43

3.2.5 Data analysis 43

3.3 Results and Discussion 44

3.3.1 Surface preparation 44

3.3.2 Validation parameters 46

3.3.2.1 Specificity 46

3.3.2.2 Range 49

3.3.2.3 Repeatability and Precision 50

3.3.2.4 Recovery and Dilution Paradox 52

3.3.2.5 Robustness: Sensitivity and Stability 55

3.3.3 Evaluation of kinetics and equilibrium contants 57

3.3.3.1

Qualitative interpretation of affinity and

kinetics

62

3.4 Conclusions 64

4 RECOVERY OF HUMAN EPIDERMAL GROWTH FACTOR

FROM Escherichia coli HOMOGENATE AND Pichia pastoris

CULTURE MEDIUM USING EXPANDED BED

ADSORPTION

4.1 Introduction 65

4.2 Materials and Methods 68

4.2.1 Materials 68

4.2.2

Optimization of adsorption and desorption using batch

binding

69

4.2.2.1 Optimization of adsorption pH 69

4.2.2.2 Optimization of elution condition 70

4.2.3 Preparation of feedstock containing hEGF 70

4.2.4 Extraction of hEGF protein 71

4.2.5 Recovery of protein by expanded bed adsorption

chromatography

71

4.2.5.1

Recovery of BSA by expanded bed

adsorption chromatography

72

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4.2.5.2 Recovery of hEGF by expanded bed

adsorption chromatography

72

4.2.6 Analytical procedures 73

4.2.6.1

Determination of hEGF concentration and

protein concentration

73

4.2.6.2 Determination of zeta potential 73

4.2.6.3 Cell Transmission Index, T 73

4.2.6.4

Sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) analysis

74

4.3 Results and Discussion 75

4.3.1 Optimization of adsorption pH 75

4.3.2 Optimization of elution conditions 76

4.3.3 Biomass-Adsorbent Interactions using Cell

Transmission Index in the expanded bed

78

4.3.4 Zeta Potential for different cells in feedstock 79

4.3.5 Recovery of BSA using expanded bed adsorption 81

4.3.6 Recovery of hEGF using expanded bed adsorption 85

4.5 Conclusions 89

5 SUMMARY, CONCLUSION AND RECOMMENDATION

FOR FUTURE RESEARCH

5.1 Summary and Conclusions 90

5.2 Suggestions for future research 92

REFERENCES 94

APPENDICES 105

BIODATA OF STUDENT 110

LIST OF PUBLICATIONS 111

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