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Manufacturing of Agarose-Based Chromatographic Media with Controlled Pore and Particle Size

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Manufacturing of Agarose-Based Chromatographic Media with Controlled Pore and Particle Size MANUFACTURING OF AGAROSE-BASED CHROMATOGRAPHIC MEDIA WITH CONTROLLED PORE AND PARTICLE SIZE by Nicolas Ioannidis A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Chemical Engineering College of Engineering and Physical Sciences The University of Birmingham 2009 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Chromatography remains the most commonly employed method for achieving high resolution separation of large-sized biomolecules, such as plasmid DNA, typically around 150-250 nm in diameter. Currently, fractionation of such entities is performed using stationary phases designed for protein purification, typically employing pore sizes of about 40 nm. This results into a severe underexploitation of the porous structure of the adsorbent as adsorption of plasmid DNA occurs almost exclusively on the outer surface of the adsorbent. In this study, the effect of two processing parameters, the ionic strength of agarose solution and quenching temperature, on the structure of the resulting particles was investigated. Three characterization methods, Atomic Force and cryo-Scanning Electron microscopy, as well as mechanical testing of single particles where used to quantify the effect of these parameters on the pore size/size distribution and mechanical properties of the adsorbent. In the presence of salt, it was found that agarose fibres tend to aggregate, leading to a gel with large pore size and wide pore size distribution. In fact, for the narrow range of ionic strength used (0-0.1m), a five-fold increase in pore size of the gel was observed. The same type of enlarged agarose structures was observed when slow cooling was applied during the gelation of agarose. The increase in pore size of the gel was also accompanied by an increase in the compression strength and the elastic modulus of the particles, i.e. particles with 200 nm pore size were found to have higher compression strength (1.5-fold difference) than those with 40 nm pore size. Abstract i ACKNOWLEDGEMENTS First of all, I would like to thank my supervisor at the University of Birmingham, Prof. A.W. Pacek, for his enormous patience and excellent guidance throughout the last four years. I would also like to thank Prof. A. Lyddiatt for his vital contribution to this work. I would like to expresses my acknowledgement to the EPSRC and Millipore who provided the funding for this work. I would also like to express my sincere gratitude to Prof. Z. Zhang, and Dr. W. Liu for providing me with Micromanipulation equipment and expertise, essential for the completion of my work. Essential for the completion of my work was also the contribution of Dr. J. Bowen in Atomic Force Microscopy, and of Dr. P. Ding in Micromanipulation. Many thanks to all the technical staff at the University of Birmingham and especially to Mr. T. Edleston, Ms. H. Jennings and Mrs. E. Mitchell for technical assistance as well as Mr. P. Stanley and Mrs. T. Morris for their assistance with Scanning Electron Microscopy Last but not least, I would like to thank Poppy for standing by me and encouraging me through this difficult time. Acknowledgements ii To my parents, Poppy, and my aunt Thalia who is no longer with us iii LIST OF ABBREVIATIONS AFM: Atomic force microscopy C–IP–I: Capture, intermediated purification, polishing CM: Carboxy methyl group DEAE: Diethylamino ethanol DNA: Deoxyribonucleic acid EBD: Electron-beam deposition HETP: Height of equivalent theoretical plate HGMF: High gradient magnetic fishing HLB: Hydrophilic-lipophilic balance HSM: High shear mixer ISEC: Inverse size exclusion chromatography MW: Molecular weight NMR: Nuclear magnetic resonance O / W: Oil-in-water pDNA: Plasmid DNA Q: Quaternary ammonium RNA: Ribonucleic acid SALS: Small angle light scattering SANS: Small angle neutron scattering SAS: Small angle scattering SAXS: Small angle neutron scattering SEC: Size exclusion chromatography SEM: Scanning electron microscopy SP: Sulfopropyl STM: Scanning tunnelling microscopy iv TBE: Tris base, boric acid and EDTA buffer solution TEM: Transmission electron microscopy UV: Ultra-violet W / O: Water-in-oil v LATIN SYMBOLS A: Constant relating to eddy diffusion and mobile phase mass transfer 3 Ad: Total area of dispersed phase (m ) 2 Amin: Minimum area occupied per molecule (m ) 2 -3 As: Specific surface area of dispersed phase (m m ) B: Constant relating to longitudinal diffusion b: Material-depended constant c.m.c: Critical micelle concentration (mol kg-1) C, C’: Constants relating to stagnant mobile phase diffusion c: Material-depended constant or surfactant concentration in the bulk (kg m-3) 0 -3 CA : Initial concentration of surfactant in the continuous phase prior emulsification (kg m ) eq -3 CA : Eq. concentration of surfactant in the continuous phase after emulsification (kg m ) Cn: Flory characteristic ratio (-) -3 Cs: Initial surfactant concentration in the emulsion (kg m ) D: Diameter (m) or diffusion coefficient (m2s-1) d32: Sauter mean diameter (µm) dmax: Particle maximum diameter (µm) dmin: Particle minimum diameter (µm) dp: Particle diameter (µm or m) E: Young’s modulus (Pa) or constant relating to adsorption kinetics F: Force (N) fHB: Correction factor g: Gravitational acceleration (m s-2) G: Shear modulus (Pa) G’: Elastic modulus (Pa) G’’: Viscous modulus (Pa) vi h: deformation (m) H: Height (m) K: Consistency index (N sn m-2) l: length of bond along the polymer backbone (nm) Lc: Column length (m) m: Meters (m) Mc: Molecular weight between crosslinks Mr: Molecular weight ms,i: Mass of surfactant adsorbed in the interface (kg) ms,t: Total mass of surfactant in emulsion (kg) N: Rounds per minute (rpm) or Avogadro’s number (-) or number of crosslinks per chain (-) n: Flow behaviour index (-) pc: Capillary pressure (Pa) r: particle radius (m) 2 1/2 (r0 ) : root-mean-square, end-to-end distance of the polymer between to crosslinks (nm) R: Gas constant (-) Re: Reynolds number (-) Rh: Hydraulic radius (m) T: Absolute temperature (°K) tbr: Breakage time (s) u: Linear velocity (m s-1) V: Volume (L or m3) v2,s: Polymer volume fraction (-) -3 Vd: Volume of dispersed phase (m ) We: Weber number (-) Y: Yield (%) zi: Number of charges in the ion (-) vii GREEK SYMBOLS α: Cone angle (°) or elongation ration of polymer chains in any direction : Shear rate (s-1) Γ: Surface excess concentration of surfactant (mol kg-1) -2 Γs: Surfactant mass surface coverage (kg m ) δgap: Rotor-stator gap (m) ε: Voidage (%) or energy dissipation rate (W kg-1) η: Apparent viscosity (Pa s) Θ: Surface coverage (-) λ: Viscosity ratio (-) µ: Viscosity (Pa s) ν: Poisson’s ratio (-) ξ: Pore size (nm) ρ: Density (kg m-3) σ: Interfacial tension (N m-1) or normal stress (Pa) φ: Dispersed phase volume fraction (-) viii TABLE OF CONTENTS INTRODUCTION i. Background 1 ii. Objectives 7 iii. Layout of the Thesis 8 CHAPTER 1: LITERATURE SURVEY 1.1. Biotechnology and downstream processing 9 1.2. Process chromatography 12 1.3. Chromatographic media 19 1.3.1. Development 19 1.3.2. General characteristics and requirements for an ideal adsorbent 25 1.3.3. Structure-diffusion relationships 29 1.4. Agarose 31 1.5. Characterization techniques 33 1.6. Methods and equipment for manufacturing agarose-based chromatographic media 46 CHAPTER 2: EXPERIMENTAL SETUP AND MATERIALS & METHODS 2.1. Manufacturing of agarose beads 54 2.1.1. Experimental rig 54 2.1.2. Materials 56 2.1.3. Agarose solution preparation 56 2.1.4. Emulsification 57 2.1.5. Centrifugation 59 2.1.6. Washing 59 2.2. Particle sizing 60 2.3. Physical properties of agarose solution and mineral oil 60 2.3.1. Viscosity measurements 60 2.3.2. Interfacial tension measurements 62 2.3.3. Density measurements 64 2.4. Microscopy 64 2.4.1. AFM sample preparation 64 2.4.2. Cryo-SEM sample preparation 65 2.4.3. Microscopy data interpretation 66 2.5. Micromanipulation 67 Table of Contents ix 2.5.1. Experimental rig and compression method 67 2.5.2. Probe manufacturing 70 2.5.3. Screen and stepper motor calibration 72 2.5.4. Force transducer calibration 73 2.5.5. Force transducer compliance 75 2.5.6. Raw micromanipulation data interpretation 77 CHAPTER 3: MANUFACTURING AND MACROSCOPIC CHARACTERIZATION OF AGAROSE BEADS 3.1. Manufacturing and macroscopic characterization 79 3.1.1. Effect of surfactant concentration and type on particle size / size distribution 81 3.1.2. Effect of ionic strength of dispersed phase on particle size / size distribution 99 3.1.3. Effect of quenching temperature of the emulsion on particle size / size distribution 102 3.1.4. Effect of impeller speed and energy dissipation rate on particle size / size distribution 103 3.2. Physical properties of agarose solution and mineral oil 109 3.2.1. Density of agarose solution and mineral oil 109 3.2.2. Interfacial tension of agarose solution and mineral oil 110 3.2.3. Rheological properties of agarose solution and mineral oil 111 CHAPTER 4: ATOMIC FORCE AND CRYO-SCANNING ELECTRON MICROSCOPY CHARACTERIZATION OF BEADED AGAROSE STRUCTURE 4.1. Pore size of agarose beads by AFM 115 4.1.1. Effect of ionic strength of agarose solution on pore size / size distribution 116 4.1.2. Effect of quenching temperature of emulsion on pore size / size distribution 126 4.1.3.
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