Electrospun Affinity Membrane for Immunoglobulin G Purification XU DAN NATIONAL UNIVERSITY OF SINGAPORE 2006 Electrospun Affinity Membrane for Immunoglobulin G Purification XU DAN （M.Eng, NUS） A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2006 ACKNOWLEDGEMENT The work presented here is the effort of a number of fantastic collaborations Without them, this thesis could never be finished More importantly, these collaborations have also made the whole graduate study extremely fruitful First, and foremost, I would like to express appreciation and gratitude to my main supervisor, Professor Seeram His infectious enthusiasm, tireless work, gentle and kindness are inspirational and make him extremely respected to work for The clarity of thought and the simplicity with which he approaches complex phenomenon serves as a constant reminder that insight occurs by asking very basic questions The influence of his wills of giving his student great freedom on project managing and personal development can not be underestimated I was extremely fortunate to have the opportunity to work with Doctor Ma Zuwei for the past two and half years, during which he has guided me in the exciting world of applied scienc He provided me with flexible and illuminating training in good experimental procedures And I appreciate his immense help, support, and catholicity throughout the project I also thank him for creating such an excellent research environment in the Nano Biochemistry Lab I would like to acknowledge all the members of the Prof Seeram’s Lab in the Division of Bioengineering whom, in one way or another, have helped to make my learning I would like to thank He Wei, Feng Yu, Bo Jun, Dr Venugopal , Satinder, Renuga, Wee Eong, Dr Kazu, Ryuji, Liu yingjun for being fantastic co-workers and colleagues who provided me with continuous support and encouragement My thanks are extended to all my classmates in the Graduate Program in Bioengineering and all my other friends at NUS I have enjoyed myself very much interacting with and learning from all of them Last but not least, my family and friends provide me with exceptional support during the whole process of my graduation study I would especially thank my best friend Junping who has been my classmate for totally seven years already for her steadfast friendship and warm-hearted tendance during the past two and a half years in Singapore Without her this process would have been immeasurably more difficult, and much less fun Finally, I would like to thank my parents, Xu Zhiyi and Lu Wen, for their unconditional love and support and their encouraging me to be curious, fearless and persistent in whatever I choose to Their immense trust to me will always be my biggest impetus TABLE OF CONTENTS ACKNOWLEDGEMENTS TABLE OF CONTENTS SUMMARY NOMENCLATURE 10 LIST OF FIGURES 12 LIST OF TABLES 15 CHAPTER 1: INTRODUCTION 1.1 Background 16 1.2 Objectives and Approach 23 CHAPTER 2: LITERATURE REVIEW 2.1 Overview 2.2 Antibody Production and Purification 2.3 26 2.2.1 Antibody Production 33 2.2.2 Antibody Purification 38 2.2.3 Affinity Chromatography 40 2.2.4 Affinity Membrane Chromatography 46 IgG and It’s Binding Properties with Protein A/G 2.4 2.5 2.3.1 Human IgG 51 2.3.2 IgG Properties and Purification 52 2.3.3 Ligands for IgG Purification 54 Affinity membrane/ligand couplings for IgG purification 60 2.4.1 Cellulose-Based Membranes 63 2.4.2 Chitin and Chitosan Based Membranes 66 2.4.3 Other Materials for Affinity Membranes 68 Market Information 70 CHAPTER 3: EXPERIMENTS 3.1 3.2 3.3 3.4 Introduction 73 3.1.1 Materials 74 3.2.2 Apparatus 74 Electrospinning of Cellulose Acetate Membrane 3.2.1 Sample Preparation for Electrospinning 76 3.2.2 Electrospinning Experiment Setup and the Process 77 CA Membrane Regeneration and Surface Modification 3.3.1 Heat Treatment 79 3.3.2 Alkali Treatment 79 3.3.3 Activation of cellulose Membrane by Sodium Periodate 79 3.3.4 BSA Immobilization Capacity of Activated Membrane 80 Ligation: Covalent Binding of Protein A/G 3.5 3.4.1 Spin Column Cartridge 82 3.4.2 Ligation of Protein A/G 82 3.4.3 Protein A/G Binding Capacity Measurement 83 Spin Column Affinity Purification Experiment 3.5.1 Experiment Preparation 84 3.5.2 Long-time Binding and Elution 84 3.5.3 Short-time Binding and Elution 85 3.6 Determination of IgG Binding Capacity 87 3.7 SDS-PAGE for Determination of the Purity of Purified IgG 87 3.8 Membrane Morphology: Microscopic and SEM Observation 90 3.9 Tensile Properties of the Single Layer Cellulose Membrane 91 3.10 XPS and FTIR Tests for Membrane Surfaces Analysis 92 CHAPTER 4: RESULTS AND DISCUSSION 4.1 Electrospinning 93 4.2 Heat Treatment 97 4.3 Alkaline Treatment 101 4.4 Oxidization 103 4.5 Protein A/G Immobilization 107 4.6 Capturing Capacity Test 109 4.7 Separation Efficiency Test 111 CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions 113 5.2 Recommendations 115 REFERENCES SUMMARY Affinity interactions can provide the very specific molecule identification which is needed for purification of important bio-molecules Although many affinity bead gels are already commercially available for protein purification, the commonly used soft gels suffer from some drawbacks for large-scale applications The performances of these gels is limited by low flow rate, weak compressibility and inevitable pore diffusion The throughput of existing gel affinity systems has often been inadequate for quick large-scale separations In recent years, micro-porous membranes have been successfully modified and various ligands have been coupled to the membrane surface for affinity chromatography purposes Using membrane filtration with affinity ligands in free solution to bind and retain specific ligates has been demonstrated to be feasible Although key factors governing the separation efficiency of these affinity systems are not yet clearly understood, many companies have put a lot of funding and energy into the affinity membrane purification industry and several products are available on the ® market now (such as Sartobind Protein A membrane serials etc.) The primary aim of our research is to use regenerated cellulose nanofiber membranes, prepared by an electrospinning method, as affinity membranes for immunoglobulin G (IgG) purification In this project, Protein A/G, which is genetically modified and specifically binds to IgG, was covalently coupled to the electrospun nanofiber membrane surface as a ligand to capture IgG molecules in free solution Both the physical and biological properties of our membrane were carefully characterized The mechanical strength of our membrane was optimized by an Instron Microtester 5848 Surface element analysis was carried out to verify the immobilization of protein A/G The amount of Protein A/G immobilized on the membrane was tested by the BCA method The membrane binding capacity of IgG was evaluated using UV absorption measurement Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis （SDS-PAGE） was carried out to evaluate the protein separation efficiency of our membrane ... A /G 2.4 2.5 2.3.1 Human IgG 51 2.3.2 IgG Properties and Purification 52 2.3.3 Ligands for IgG Purification 54 Affinity membrane/ ligand couplings for IgG purification 60 2.4.1 Cellulose-Based Membranes.. .Electrospun Affinity Membrane for Immunoglobulin G Purification XU DAN （M.Eng, NUS） A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN BIOENGINEERING NATIONAL... Dimethylformamide 97 DSC Differential Scanning Calorimeter 20 GF Gel Filtration 20 HIC Hydrophobic Interaction Chromatography 20 IEX Ion Exchange Chromatography IgG Immunoglobulin G 53 IVIG Intravenous