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Handbooks in Separation Science Series
Ion-Exchange Chromatography and Related Techniques
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Contents
Contributors
1. Concepts and milestones in the development of ion-exchange chromatography
1. Introduction
2. Fundamentals
2.1. Retention mechanism for small ions
2.2. Retention mechanisms for polyelectrolytes
3. Column chromatography
3.1. Porous polymer ion exchangers for the separation of low-mass ions
3.2. Restricted access media
3.3. Ion exchangers for large-scale separations
4. Large-scale ion-exchange separations
5. Landmark developments in biotechnology for downstream processing using ion-exchange chromatography
References
2. Equilibria and kinetics of ion-exchange of biopolymers
1. Introduction
2. Dynamic models
2.1. Classification of the models
2.2. Formulation of mass balance equations in the GR model
2.3. Transport-dispersive model
2.3.1. Model formulation
2.4. Compatibility of GR and TD models
2.5. Reaction-dispersive model
3. Kinetic equations of adsorption-desorption rate
3.1. Kinetics of SMA formalism
3.2. Kinetics of cooperative adsorption
3.3. Kinetics of protein unfolding upon adsorption
4. Adsorption-desorption equilibria: Isotherm equations
4.1. SMA formalism
4.2. Cooperative adsorption isotherm
4.3. CPA isotherm
4.4. Determination of isotherm coefficients
4.4.1. SMA model
4.4.2. Cooperative adsorption isotherm
4.4.3. CPA isotherm
5. Causes of misinterpretation of the elution data
5.1. Effect of feed viscosity on the process kinetics
5.2. Effect of competitive adsorption
5.3. Effect of column void volumes
6. Procedure for design of IEX process
References
3. Stationary phases for ion separations
1. Ion-exchange terminology
2. Classification of ion-exchangers
2.1. Matrix or type of substrate material
2.1.1. Inorganic materials
2.1.2. Synthetic organic polymers
2.1.3. Hybrid matrices
2.2. Structure of ion-exchangers
2.2.1. Column packing morphology
2.2.2. Localization of fixed charges in ion-exchangers
Ionogenic groups distributed in a whole volume of particle
Controlled porosity particles or superficially porous ion-exchangers
Electrostatically agglomerated ion-exchangers
Immobilized ionogenic polymer layers
Encapsulated ion-exchangers
Ion-exchangers coated with an oppositely charged polymer
Covalent bonding or grafting of a polymer layer to an activated substrate surface
Isolated ionogenic groups on substrate surfaces, or chemically modified substrates
2.3. Types of functional groups
2.3.1. Positively charged functional groups (anion-exchangers)
2.3.2. Negatively charged groups (cation-exchangers)
2.3.3. Zwitterionic and polyampholyte ion-exchangers
2.3.4. Complexing ion-exchangers
2.4. Ion-exchange capacity
References
4. Stationary phases for the separation of biopolymers by ion-exchange chromatography
1. Introduction
2. Uniform agarose-based ion-exchange chromatographic media
3. Gigaporous ion-exchange chromatographic media
3.1. Gigaporous PSt-based ion-exchange chromatographic media
3.2. Gigaporous PGMA-based ion-exchange chromatographic media
3.3. DEAE macroporous agarose chromatographic media
3.4. CM macroporous agarose chromatographic media
4. Other ion-exchange stationary phases for bioseparations
4.1. Monolithic columns
4.2. Membrane chromatography
4.3. Cryogels
4.4. Mixed-mode chromatography
5. Summary and outlook
References
5. Ion-exchange separations of biomacromolecules on grafted and surface-modified polymers
1. Introduction
2. Stationary phases
2.1. Design of polymer-functionalized ion exchangers
2.2. Introduction of the surface polyelectrolytes and their modification
2.3. Typical commercial stationary phases
3. Adsorption and uptake theory
3.1. Three-dimensional adsorption
3.2. Facilitated mass transfer by chain delivery effect
4. Applications
4.1. Features of practical applications
4.2. Application examples
References
6. Extraction chromatography of actinides
1. Introduction
2. Extractants for actinide separation
3. Ligand impregnated resins for actinides
3.1. Monoamide impregnated resins
3.2. Malonamide impregnated resins
3.3. Diglycolamide impregnated resins
3.4. Multiple DGA impregnated resins
4. Room temperature ionic liquids in extraction chromatography
4.1. TODGA/RTIL resin
4.2. C4DGA and T-DGA/RTIL resins
5. Ligand grafted resins for actinides
5.1. Monoamide grafted resins
5.2. Malonamide grafted resins
5.3. Diglycolamide grafted resins
6. Composite beads for extraction chromatography
7. Perspectives
Abbreviations
References
7. Ion-exchange membrane chromatography
1. Introduction
2. Transport phenomena in membrane chromatography
3. Module design
4. Promising ion-exchange membranes for bioseparations
5. Conclusions
References
8. Ion-exclusion chromatography
1. Principle
2. Apparatus
3. Ion-exchange resin columns used in ICE
4. Eluent conditions
5. Detection methods
5.1. Conductivity detection
5.1.1. Direct detection
5.1.2. Enhancement of conductivity by postcolumn reaction
5.2. UV-VIS detection
5.2.1. Direct UV detection
5.2.2. Postcolumn derivatization
5.3. Mass spectrometry
5.4. Charged aerosol detector
6. Separations of nonionized substances
7. Separation of ammonium and amines
8. Vacancy ion-exclusion chromatography
9. Ion-exclusion/cation-exchange chromatography
10. Ion-exclusion/anion-exchange chromatography
Abbreviations
References
9. Chelation ion chromatography
1. Introduction
2. Theoretical aspects of complexation in liquid chromatography
2.1. Complexation in the mobile phase
2.2. Complexation in the stationary phase
3. Ion-exchange chromatography with the complex formation in the mobile phase
3.1. Cation-exchange chromatography with complexing eluents
3.1.1. Fixed-site cation-exchangers and complexing eluents
3.1.2. Dynamically modified cation-exchangers and impregnated adsorbents
3.1.3. Ion-pair chromatography of complexed metal ions
3.2. Anion-exchange chromatography
3.2.1. Fixed-site anion-exchangers and complexing eluents
3.2.2. Dynamically modified anion-exchangers and ion-pair mode
4. Chelating phases for ion-exchange chromatography
5. Application areas of chelating ion-exchangers
Abbreviations
References
10. Displacement chromatography with ion-exchangers
1. Principles of displacement chromatography
1.1. Basic concepts of displacement chromatography
1.2. Variant forms of displacement chromatography
1.2.1. Selective displacement chromatography
1.2.2. Sample displacement chromatography
1.2.3. Complex displacement chromatography
1.3. Theoretical models for displacement chromatography
2. Ion-exchange displacers
2.1. Displacers for ion-exchange chromatography
2.2. Approaches for displacer screening and design
3. Applications of ion-exchange displacement chromatography
3.1. Displacer chromatography process development and optimization
3.2. Applications
3.2.1. Displacement chromatography for the purification of recombinant proteins
3.2.2. Displacement chromatography for proteomic analysis
3.2.3. Applications of sample displacement chromatography
4. Prospects and outlook
References
11. Instrumentation for ion chromatography
1. Solvent delivery systems for IC applications
1.1. High-pressure piston pump
1.2. Eluent production modules
2. Detectors for IC
2.1. Conductivity detection
2.1.1. Suppressors for suppressed conductometry
Column-type suppressors
The membrane type suppressors
2.1.2. Charge detector
2.1.3. Direct conductometry (nonsuppressed conductometry)
2.2. Electrochemical detection
2.3. Photometric detection
2.4. Postcolumn reaction system
2.5. Mass spectrometry detection
2.6. Multiple detections
3. Injection system
3.1. Injection valve with sample loop
3.2. Preconcentration
4. Column oven
5. Column hardware
References
12. Instrument platforms for large-scale ion-exchange separations of biomolecules
1. Introduction
2. Chromatography columns
3. Ion exchange matrices
3.1. Process steps in ion-exchange chromatography
4. Chromatography equipment
5. Scale-up of ion-exchange processes
5.1. Understanding the product and resin selection
5.1.1. Column design and size
5.1.2. Process parameters
5.1.3. Validation and cleaning
5.1.4. Equipment and facility consideration
5.1.5. Mode of operations of IEC
5.2. Necessary calculations for IEC scale-up
5.3. Common problems associated with IEC scale-up from lab to manufacturing scale
5.3.1. Pressure drop
5.3.2. Buffer preparation at a manufacturing scale
5.3.3. Column packing and cleaning
5.3.4. Validation of a scaled-up process
References
13. Method development for large molecules IEX separations
1. Introduction
2. Column, stationary phase, and instrumentation considerations
2.1. Stationary phase characteristics
2.2. Column dimensions
2.3. Column hardware and instrumentation
2.4. Detection
3. Elution modes
3.1. Salt gradient mode
3.1.1. General considerations
3.1.2. Practical considerations for salt-gradient separations
Mobile phase pH and buffer
Salt additive in the mobile phase
3.2. pH gradient mode
3.2.1. General considerations
3.2.2. Practical considerations for pH gradient separations
Mobile phase buffers
The effect of the column on pH of the effluent
Empirical correction of nonlinear pH gradients
3.3. Salt-mediated pH gradient mode
4. IEX mode for nucleic acid separations
4.1. General considerations
4.2. Practical considerations
4.3. Denaturing elution conditions for nucleic acid analysis
5. On-off mechanism of retention
5.1. Multisegmented gradients (multiisocratic elution) vs linear gradients
6. Need for IEX platform methods
7. Systematic method development
7.1. Screening experiments
7.2. Method optimization in general
8. Practical advice for systematic method optimization
8.1. Method optimization in salt gradient mode
8.2. Method optimization in pH gradient mode
8.3. Method optimization in salt mediated pH gradient mode
8.4. Method optimization in ion-pairing IEX
9. Perspectives
Declaration of competing interest
References
14. Sample preparation for ion-exchange separations
1. Introduction
2. Liquid-phase extraction
2.1. Solid samples
2.2. Gas phase samples
2.3. Liquid-liquid extraction
3. Solid-phase extraction
3.1. Inorganic ions (matrix removal and concentration)
3.2. Inorganic ions (speciation)
3.3. Inorganic ions (extraction chromatography)
3.4. Organic ions
3.5. Biomacromolecules
3.6. Microextraction formats
4. Membrane-based extraction
4.1. Electrodialysis
4.2. Electromembrane extraction
References
15. Separation of ions by ion chromatography
1. Anion-exchange chromatography
1.1. Eluents
1.2. Stationary phases
1.3. Separation of anions
2. Cation-exchange chromatography
2.1. Stationary phases
2.2. Separation of cations
References
16. Applications of ion chromatography in environmental analysis
1. Introduction
2. Ion chromatography and related techniques
2.1. Water samples
2.2. Atmospheric samples
2.3. Solid samples
3. IC development perspectives
References
17. Applications of ion chromatography in food analysis
1. Introduction
2. Inorganic anions
3. Inorganic cations
4. Carbohydrates
5. Organic acids
6. Food safety issues
References
18. Separation of saccharides by ion-exchange chromatography
1. Introduction
2. Separation of saccharides by HPAEC
2.1. Monosaccharide and disaccharides analysis
2.2. Oligosaccharides and polysaccharides separation
2.3. Glycoconjugate separation
3. Separation of saccharides by ligand exchange chromatography
3.1. Monosaccharide and disaccharides separations
3.2. Oligosaccharides separation
4. Ion exclusion chromatography
5. Ion-pair chromatography
5.1. Monosaccharides and disaccharides separations
References
19. Separation of oligonucleotides by ion-exchange chromatography
1. Introduction
2. Column packings for the ion-exchange separations
3. Nucleotides and sugar nucleotides
3.1. Nucleotide sugars
4. Oligonucleotides
5. Phosphorothioated oligonucleotides
6. Mixed mode stationary phases
7. Multidimensional chromatography
8. Purification of oligonucleotides
References
20. Separation of oligonucleotides by ion-exchange and ion-pair chromatography
1. Introduction
2. Types of oligonucleotides
3. Endogenous RNAs
3.1. Messenger RNA
3.2. Ribosomal RNA
3.3. Transfer RNA
3.4. Small nuclear RNA
3.5. Small nucleolar RNA
3.6. Micro RNA
3.7. Long noncoding RNAs
4. Therapeutic oligonucleotides
4.1. Small interfering RNA
4.2. Antisense oligonucleotides
4.3. Aptamers
4.4. mRNA therapeutics
4.5. Single guide RNA
5. Charged-based separations of oligonucleotides
5.1. Ion-exchange chromatography
5.2. Ion-pair chromatography
5.3. Mixed-mode chromatography
6. Nonspecific adsorption
7. Sample preparation for oligonucleotides
8. Liquid chromatography-mass spectrometry of oligonucleotides
8.1. Two-dimensional liquid chromatography separations
9. Selected applications of ion-exchange and ion-pair chromatography to the determination of oligonucleotides
9.1. IEC of siRNAs
9.2. IEC of an aptamer
9.3. IPC of an antisense therapeutic
9.4. Two-dimensional chromatographic approaches for impurity determinations
9.5. Two-dimensional chromatographic separation of an mRNA digest
10. Future directions
References
21. Separation of proteins by ion-exchange chromatography
1. Introduction
2. Adsorption: Principles and models
3. Evolution of resins
4. Investigational approaches
5. Common modes of operation
6. Perspectives
References
22. Separation of proteins by mixed-mode chromatography
1. Introduction
2. Mixed mode ligands
3. Effect of salt and pH on mixed-mode chromatography
4. The third dimension
5. High-throughput screening of mixed-mode resins
6. Monoclonal antibody purification
7. Conclusion
References
23. Process modeling of protein separations by ion-exchange chromatography
1. Introduction
2. Mechanistic modeling of chromatography
2.1. Mechanistic model equations and zone movement in the column
3. Plate height and related variables in isocratic elution
4. Resolution Rs in isocratic elution
5. Ion-exchange equilibria
6. Linear gradient elution
6.1. Retention in linear gradient elution
6.2. Peak width, HETP, and Rs in linear gradient elution
6.3. Iso-resolution curves in linear gradient elution
7. Applications of the mechanistic models and the simplified methods
7.1. Stepwise-elution process design based on linear gradient elution data
7.2. Flow-through chromatography
7.2.1. Process design
7.3. Model simulations for flow-through chromatography
7.4. Summary
8. Capture process design
8.1. Multicolumn periodic counter current operation
8.2. Flow velocity gradient loading operation
8.3. Model simulations
8.4. Summary
References
24. Applications of ion-exchange chromatography for the purification of antibodies
1. Antibodies: Introduction, recombinant expression, manufacturing, and purification
1.1. Monoclonal antibodies and variant forms
1.2. Antibody expression, manufacturing, and purification
1.2.1. Recombinant expression and manufacturing
1.2.2. Purification workflow
Antibody purification workflow
2. Cation-exchange chromatography of antibodies
2.1. Principles and goal
2.2. Starting conditions and parameters for optimization
2.2.1. Resin screening
2.2.2. Elution program development
2.2.3. Optimization of loading condition
2.2.4. Determination of operating space
2.3. Case studies
3. Anion-exchange chromatography of antibodies
3.1. Principles and goal
3.2. Starting conditions and parameters for optimization
3.3. Case studies
4. Commercial ion-exchange resins
4.1. Ion-exchange ligands, linking chemistries, and backbone materials
4.2. Commercial ion-exchange resins
5. Scale-up and scale-down of ion-exchange chromatography
6. Tips and tricks
Acknowledgment
References
25. Conformational changes of biomolecules in ion-exchange chromatography
1. Introduction
2. Mechanisms
2.1. Interconversion between different conformational states
2.2. Unfolding and aggregation
3. Detection of protein conformational changes
3.1. Spectroscopic methods
3.2. Differential scanning fluorimetry (DSF) and differential scanning calorimetry (DSC)
3.3. Hydrogen/deuterium exchange (HDX)
4. Key contributing factors
4.1. Protein intrinsic stability
4.2. Stationary phase properties
4.2.1. Ligand
Ligand type
Ligand density
4.2.2. Particle morphology
Pore size distribution (PSD)
Polymer-grafted resins
4.2.3. Surface property (hydrophobicity)
4.3. Practical implications
4.3.1. Mass load
4.3.2. Contact time (hold step)
4.3.3. Operating flow rates
4.3.4. Operating temperature
4.3.5. Mobile phase pH
4.3.6. Salt and excipients
References
26. Continuous ion-exchange chromatography for protein polishing and enrichment
1. Introduction
2. Limits of batch chromatography
3. Continuous chromatography for protein purification
3.1. Rotating chromatography
3.1.1. Column switching chromatography
3.1.2. Annular chromatography
3.2. Simulated moving bed
3.2.1. Classical simulated moving bed
3.2.2. Gradient simulated moving bed
3.2.3. Simulated moving bed for ternary separation
3.3. Multicolumn continuous chromatography
3.3.1. Multicolumn counter-current solvent gradient purification
3.3.2. Two-column batch-to-batch recirculation process
3.3.3. N-rich
3.3.4. Flow2
3.3.5. Multicolumn self-displacement chromatography
3.4. Comparison of different continuous modes
4. Process design for continuous purification
4.1. Experiment-based design
4.2. Model-based design
5. Applications for protein polishing using ion-exchange
5.1. Monoclonal antibodies
5.2. PEGylated proteins
6. Conclusion
References
27. Separation of bio-particles by ion-exchange chromatography
1. Introduction
2. The principles of separating bio-particles by IEC
3. Challenges and coping strategies for separating bio-particles by IEC
3.1. Limitations of low binding capacity and coping strategies
3.2. Time-consuming issue and coping strategies
3.3. Low recovery and coping strategies
3.3.1. Effects of media pore size on recovery
3.3.2. Effects of ligand density on recovery
3.3.3. Effects of mobile phase on recovery
4. Design of IEC-based bio-particle separations
4.1. Special considerations
4.1.1. Expression systems
4.1.2. The source of impurities
4.1.3. Flow-through mode
4.1.4. Separation stages
4.1.5. Salts in the mobile phase
4.2. Representative examples of bio-particle separations by IEC
4.2.1. Separation of HBsAg-VLPs
4.2.2. Separation of the influenza virus
4.2.3. Separation of AAV Serotype 9
4.2.4. Separation of EVs
5. Future prospects
Abbreviations
Acknowledgment
References
28. Virus removal in bioprocessing using charged media
1. Introduction
2. Virus physicochemical properties
3. Protein interference
4. Chromatography format
5. Cation exchange and multimodal ligands
6. Conclusions
References
29. Role of temperature in ion-exchange processes of separation and purification
1. Influence of temperature on the stability of ion-exchange resins
2. Influence of temperature on the kinetics and dynamics of ion exchange
3. Influence of temperature on the ion-exchange equilibrium
4. The role of temperature in traditional ion-exchange processes using auxiliary reagents
4.1. Reagentless separation processes
References
Index
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