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Bioseparations Engineering

Bioseparations Engineering - 01 edition

ISBN13: 978-0471244769

Cover of Bioseparations Engineering 01 (ISBN 978-0471244769)
ISBN13: 978-0471244769
ISBN10: 0471244767
Cover type:
Edition/Copyright: 01
Publisher: John Wiley & Sons, Inc.
Published: 2001
International: No

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Bioseparations Engineering - 01 edition

ISBN13: 978-0471244769

Michael R. Ladisch

ISBN13: 978-0471244769
ISBN10: 0471244767
Cover type:
Edition/Copyright: 01
Publisher: John Wiley & Sons, Inc.

Published: 2001
International: No
Summary

Multidisciplinary resource for graduate studies and the biotechnology industry

Knowledge of the genetic basis of biological functioning continues to grow at an astronomical rate, as do the challenges and opportunities of applying this information to the production of therapeutic compounds, specialty biochemicals, functional food ingredients, environmentally friendly biocatalysts, and new bioproducts from renewable resources. While genetic engineering of living organisms transforms the science of genomics into treatments for cancer, diabetes, and heart disease, or products for industry and agriculture, the science and technology of bioseparations are the keys to delivering these products in a purified form suitable for use by people.

The methods, theory, and materials that reduce the science of bioseparations to practice, whether in the laboratory or the plant, are the subjects of Bioseparations Engineering. Examples address purification of biomolecules ranging from recombinant proteins to gene therapy products, with footnotes detailing economics of the products. Mechanistic analysis and engineering design methods are given for:

  • Isocratic and gradient chromatography
  • Sedimentation, centrifugation, and filtration
  • Membrane systems
  • Precipitation and crystallization

Topics addressed within this framework are: stationary phase selection; separations development; modeling of ion exchange, size exclusion, reversed phase, hydrophobic interaction, and affinity chromatography; the impact of regulatory issues on chromatography process design; organization of separation strategies into logical sequences of purification steps; and bridges between molecular biology, combinatorial methods, and separations science.

A result of teaching and developing the subject matter over ten years, Bioseparations Engineering is an ideal text for graduate students, as well as a timely desk book for process engineers, process scientists, researchers, and research associates in the pharmaceutical, food, and life sciences industries.

Author Bio

Ladisch, Michael R. : Purdue University

Michael R. Ladisch, Phd, is Distinguished Professor in the Department of Agricultural and Biological Engineering and the Department of Biomedical Engineering, and the Director of the Laboratory of Renewable Resources Engineering, at Purdue University in West Lafayette, Indiana.

Table of Contents

Preface.
Acknowledgements.

Bioseparations.
1.0 Introduction.
1.1 The Manner in Which the Bioproduct Is Associated With the Cell or Organism Defines Its Initial Recovery Characteristics.
1.2 Physical Processing and Water Removal Steps Are Important Separation Methods for Large Volume Products.
1.3 Minimal Downstream Processing is Characteristic of Some Large Volume Extracellular Enzyme Products Used in the Food and Textile Industries.
1.4 There Are Three Major Categories of Bioproducts: Cells, Intracellular Products and Extracellular Products.
1.5 Recombinant Proteins Derived from E. coli Are Initially in the Form of Insoluble, Intracellular Inclusion Bodies.
1.6 The Unit Operations of Bioseparations Are Grouped Into Five Major Categories.
1.7 Bioinformatics Will Lead to Products Which Bioseparation Methods Must Purify.
1.8 Bioseparations Engineering Plays a Major Role In The Successful Development of Bioprocesses For The Manufacture of Therapeutic Molecules and Specialty Chemicals.

Sedimentation, Centrifugation, and Filtration.
2.0 Introduction.
2.1 Solid/Liquid Separations by Sedimentation or Centrifugation are Based on Differences in Particle Size and Density.
2.2 Centrifugation Uses Mechanical Force to Amplify the Differences in Size and Density Between Wet Biological Materials and the Aqueous Media In Which the Solids are Found.
2.3 The Volumetric Rate of Clarified Supernatant is Maximized by a Large Density Difference and Low Viscosity.
2.4 Centrifuge Speed Is Limited By Stress In the Bowl's Wall and By Its Materials of Construction.
2.5 The Disc Stack Centrifuge Enables Continuous and Rapid Processing of Cell and Colloidal Suspensions.
2.6 A Decanter Centrifuge Is Less Efficient for Recovery of Micro-organisms Than a Disc Centrifuge.
2.7 Sterility, Containment and Heat from Mechanical Work Affect Design of Process Centrifuges.
2.8 Centrifuge Design for Biotechnology Processes Incorporates Cleaning-in-Place and Sterilization Capabilities.
2.9 Centrifuge Containment Is Necessary For Processing of Some Types of Biotechnology Products.
2.10 Filtration.
2.11 A Fluid's Superficial Velocity, or Flux, Through Filter Cake Is Proportional to A Permeability Coefficient Expressed in Units of Darcies.
2.12 Diatomaceous Earth and Perlites (Volcanic Rock) Serve to Enhance Permeability of Filter Cakes and Aid Filtration of Fermentation Broths.
2.13 Filtration for Streptomycin Recovery Requires Coagulation of the Mycelia and Addition of A Filter Aid.
2.14 Rotary Vacuum Filters Enable Continuous Filtration of Bioproducts Generated in Large Volumes of Fermentation Broth.
2.15 Penicillin G from Penicillium chrysogenum is Recovered by Rotary Filters Prior to Its Hydrolysis by Immobilized Penicillin Acylase.

Membrane Separations.
3.0 Introduction.
3.1 Microfiltration Membranes Remove Particles Whose Sizes Range from 0.1 to 10 Microns.
3.2 Molecular Filtration by Ultrafiltration and Reverse Osmosis Utilizes Supported Membranes with Nanometer Sized Pores.
3.3 The Flux, jv, Through A Membrane Follows Darcy's Law When the Osmotic Pressure Difference Across the Membrane Is Small.
3.4 The Gibbs and van't Hoff Equations Provide A Basis For Calculating Estimates of Osmotic Pressures.
3.5 Dissociation of Salts in Aqueous Solutions Increases Osmotic Pressure.
3.6 Concentration Polarization Reduces Flux.
3.7 Flux Increases With Increasing Temperature and Fluid Velocity Across the Membrane's Surface.
3.8 Pore Occlusion And Concentration Polarization Can Be More Important Than Osmotic Pressure in Determining Flux For Membrane Filtration of Proteins.
3.9 Flux Equations Are Classified Into (i) Osmotic Pressure Dependent, (ii) Hydraulic Pressure Dependent, and (iii) Pressure Independent (Concentration Polarization) Regimes.
3.10 Dimensional Analysis of Momentum and Diffusive Transport Processes Enables Estimation of Flux From Membrane and Fluid Properties.
3.11 Solute Flux in Dialysis Is Based on A Concentration Gradient, Not A Pressure Gradient.
Engineering Concepts of Membrane Applications.
3.12 Membrane Separations of Small Particles Utilize Fibrous or Particulate Depth Filters And Isotropic (Symmetric) Screen Filters.
3.13 Membranes Are Packaged in Flat Sheet or Hollow Fiber Cartridge Configurations.
3.14 The Pressure At Which Gas Flows Through a Wetted Membrane Gives A Measure of Its Pore Size (Bubble Point Test).
3.15 Sterilization of Human Plasma Proteins, Harvesting of Recombinant Microbial Cells, and Recovery of Cell Culture Products Are Applications of Microfiltration.
3.16 Aggregation of Proteins Promotes Membrane Fouling And Decreases Flux.
3.17 Dialysis and Evaporation or Reverse Osmosis Processes Remove Ethanol From Beer to Yield Beverages With Alcohol Content Reduced.

Precipitation Crystallization, and Extraction.
4.0 Introduction.
4.1 Addition of Neutral Salts, or An Acid or Base to Aqueous Solutions Induces the Solute to Precipitate.
4.2 Alcohols Decrease Solvating Power of Water by Lowering the Dielectric Constant of the Solution.
4.3 Neutral Salts Added to Solutions of Amino Acids or Proteins Cause Precipitation By Hydrophobic Interactions.
4.4 The Logrithmic Decrease in Amino Acid and Protein Solubility is Proportional to Increasing Salt Concentration (Cohn's Equation).
4.5 The Separation Factor For Two Proteins, Lysozyme and a-Chymotrypsin, Is Calculated From Their Distribution Coefficients (An Example).
4.6 Fractionation of Two Proteins By Precipitation Requires That Their Solubilities Are Significantly Different From Each Other.
4.7 pH, Temperature, and Initial Concentration Also Affect Protein Solubility.
4.8 Heat of Solution Effects Can Be Significant for Proteins.
4.9 The Salting-Out Constant, Ks, Combines Salting-Out and Salting-In Effects Which Characterize Hydrophobic Interactions.
4.10 Hydrophobic Contact Areas F of Selected Proteins, Ranging From 20 to 42% of Total Surface Area, May Be Determined From Ks.
4.11 Ks May Be Calculated From The Protein's Dipole Moment (?), Contact Area (F), And Surface Tension Increment (s) (Ovalbumin Example).
4.12 Graphing of Ks vs. Molal Surface Tension Increment (s) Enables Calculation of Protein Solubility in Different Salt Solutions (An Example).
4.13 Thermodynamics Offer An Explanation For Both Salting-in and Salting-out Effects.
4.14 Protein Micelles in Milk Precipitate by Enzyme Induced Coagulation.
Crystallization.
4.15 A Nucleus or Critical Cluster Known As An Embryo Is Required For Crystallization.
4.16 The Analysis of a Crystallization Processes is Based on Differences in Chemical Potential and the Saturation Ratio.
4.17 The Work For Forming An Embryo Is Associated With Building the Surface of a Crystal And Increasing Its Volume.
4.18 The Diameter of An Embryo At Equilibrium Represents The Critical Diameter At Which Crystallization May Be Induced.
4.19 The Work Function Is A Thermodynamic Expression That Represents A Surface Tension and Temperature Dependent Activation Energy For Homogeneous Nucleation.
4.20 The Induction Period For Crystallization Is Proportional to the Cube of Surface Tension (g3) and Inversely Proportional to (ln S)2.
4.21 Surface Tension May Be Estimated From the Ratio of Solute Concentration In The Crystal to Its Concentration In Solution.
4.22 Patterns of Particle Accumulation As A Function of Solute Concentration Sometimes Distinguish Heterogeneous From Homogeneous Nucleation (Cholesterol and Citric Acid Examples).
4.23 The Transition From Heterogeneous Nucleation at Low Super-saturation to Homogeneous Nucleation At High Supersaturation Facilitates Estimates of Interfacial Surface Tension.
4.24 Heterogeneous Nucleation Can Give Rise to Anomalies Upon Scale-up.
4.25 Miers Plots Represent Crystallization Paths For Solutions Brought To Supersaturation By Cooling And Through Solvent Removal By Evaporation.
4.26 Numerical Solutions of Material Balances Give Curves That Represent The Decrease In Solute As A Function of Time For Heterogeneous Crystallization.
Leaching and Extraction.
4.27 "Leaching Is the Preferential Solution of One or More Constituents of A Solid Mixture By Contact With A Liquid Solvent (Terybal, 1968)".
4.28 Supercritical Carbon Dioxide Is An Effective Extractantr Solid Bioproducts.

Principles of Liquid Chromatography.
5.0 Introduction.
5.1 Liquid Chromatography Systems Are Classified by Pressures That Characterize Their Operation (HPLC, LPLC, and MPLC).
5.2 This Chapter Presents The Principles And Practices of Analyzing and Scaling-up Chromatography Column Performance from Experimental Measurements.
5.3 Liquid Chromatography Systems Consist of Columns, Injectors, Detectors, Pumps, Fraction Collectors, and Stationary and Mobile Phases.
5.4 The Target Molecule Is The Molecule Which Is To Be Recovered In A Purified Form.
5.5 The Nomenclature of Chromatography Is Summarized In Schematic Diagrams.
5.6 Gradient Chromatography Is A Form of Adsorption.
5.7 Gradients Are Formed By Combining Two or More Liquid Buffers to Give A Time-Varying Change in Displacer Concentration.
5.8 Liquid Chromatography Columns Are Packed Using Liquid Slurries.
5.9 Some Types of Stationary Phases Undergo Significant Swelling When Hydrated In Water or Buffer.
5.10 Convective Flow Through Gigaporous Particles With Transecting Pores May Occur At High Pressures.
5.11 Pellicular Particles, Polymer Monoliths, Rolled Stationary Phases, and Bundles of Hollow Fibers, Represent Other Forms of Stationary Phases.
5.12 Plate Count or Plate Height (H.E.T.P.) Gives A First Indication of Packing Efficiency.
5.13 Poisson and Gaussian Distribution Equations May be Used to Calculate Elution Profiles of Single Chromatography Peaks.
5.14 Many Peaks That Elute From Chromatography Columns Are Skewed Due to Intracolumn And Extra Column Dispersion Effects (Exponentially Modified Gaussian Peaks).
5.15 One-dimensional Model of Differential Chromatography Enables Simulation of Elution Profiles.
5.16 Sample (Feed) Volumes Affect the Calculation of Plate Count Due to Contributions of the Feed Volume to Peak Width.
5.17 Contributions to Peak Broadening Due to Particle Size and Flowrate Effects Are Given by the van Deemter Equation (Grushka, et al., 1975; van Deemter, et al., 1956).
5.18 The Effect of Particle Size, Flowrate, Solute, and Temperature on Plate Height Is Modeled Using Dimensionless Numbers Re, Sc, Pe, Nu, and Da (Derivation of Athalye, Lightfoot et al.).
5.19 Mass Transfer and Adsorption Kinetics Also Impact Plate Height.
5.20 Chromatographic Capacity Factors Are Determined from Peak Retention.
5.21 Chromatographic Separations Are Defined By Divergence of Peak Centers (Capacity Factors, Phase Ratios, and Resolution).

Liquid Chromatography Scale-up.
6.0 Introduction.
Linear Chromatography.
6.1 Scale-up Rules Enable Initial Specification of Chromatography Columns.
6.2 Scale-up Rules for Size Exclusion Chromatography (SEC) Assume Pore Diffusion Controls.
6.3 Mass Transfer Can Be a Limiting Factor at Slow Flowrates, or For Solutes That Have Slow Diffusion Rates.
6.4 Scale-up Rules Are Similar For Pore Diffusion and Mass Transfer Limiting Cases.
6.5 Scale-up When Mass Transfer and Pore Diffusion are of Comparable Magnitude Requires Combination of These Resistances.
6.6 A Material Balance Combined With Plate Count Facilitates Simulation of Elution Profiles for Linear Equilibrium: Size Exclusion and Ion Exclusion Examples (with contributions by Scott Rudge).
6.7 Physical Properties of Stationary Phase, Mobile Phase, and Feed Sample Should Not Be Forgotten When Analyzing Column Performance.
6.8 Ion Exclusion Has Possible Applications for a Greener Chemical Industry.
6.9 Linear Chromatography May Depend on Particle Size, Temperature, or Solute Concentration Effects (Case Study for Linear Chromatography Scale-up).
6.10 The Craig Model May Be Used to Predict Elution Profiles For Retained Components (k? > > 0) (with contributions by Ajoy Velayudhan).
6.11 The Stirred-Tank-In-Series Model of Chromatography Is Based on A Material Balance (k? ? 0) (with contributions by Ajoy Velayudhan).
6.12 The Craig and Stirred Tank in Series Models Give Similar Results (Example: Glucose/Fructose Separation).
Non-Linear Chromatography.
6.13 Local Equilibrium Theory Relates Elution Profiles For An Adsorbing or Desorbing Solute To Its Equilibrium Isotherm (with contributions by Juan Hong).
6.14 Desorption Isotherms May Differ From Adsorption Isotherms (Hysteresis Effects and Local Equilibrium Theory).
6.15 Triangular Peaks Are Associated With Non-Linear Chromatography (Overload Conditions).
6.16 Equilibrium and Mass Transfer Theories Can be Used to Calculate the Shape of The Front of A Single Peak For Non-Linear Chromatography.
6.17 Scale-up of Non-Linear Chromatography is Based on Maintaining the Relative Peak Position and Overlap Two or More Peaks at a Fixed Ratio.
6.18 Ratios of the Width of Mass Transfer Zones at Process and Bench Scales Are The Basis of Scale-up for Non-Linear Chromatography.
6.19 Batch Equilibrium Experiments Are Needed For Determining Non-Linear Equilibria or Confirming Equilibrium Constants Obtained from Column Chromatography Measurements.
6.20 A Competition Factor In the Langmuir Equation Accounts For Cases Where Adsorption of One Solute Affects The Other.
6.21 A Difference in Equilibrium Curves of Two Components Indicates That Separation Is Possible (Langmuir Isotherm).
6.22 Differences in Rates of Adsorption May Enable a Separation To Be Achieved When the Equilibrium Isotherms for Two Components are Similar.
Hydrodynamics.
6.23 Compression of Gel-Type Stationary Phases in Packed Beds May Cause Increased Pressure Drops (Case Study for Styrene/DVB Gel Type Ion Exchanger).
6.24 Column to Particle Diameter Should Exceed 80 In Order to Minimize Dispersion by Fingering.
6.25 Mixing And Dead Volumes Must Be Minimized In Liquid Chromatography Systems (Fittings, Injectors, Tubing, and Feed Distributors).
Productivity and Costs.
6.26 The Mobile Phase Is A Major Operational Cost for Process Liquid Chromatography (WFI Water and Other Solvents).
6.27 Special Operating Protocols Are Required For Storing the Mobile Phase Until It Is Used.
6.28 Process Hygiene Affects Choice of Materials of Construction for Column Components (Use of NaOH for Cleaning-in-Place).
6.29 The Stationary Phase is The Single Most Important Factor For Purification Development.
6.30 Yield Represents Product Recovery Regardless of Its Extent of Purification.
6.31 The Productivity, Pprod, of A Column for Each Cycle Depends on The Acceptable Extent of Purification of the Product.
6.32 The Calculation of Costs Is Based on The Productivity of The Separation.
6.33 Recycle and Moving "Stationary" Phase Chromatography Increase Productivity.
6.34 A Moving Bed System Moves the "Stationary" Phase to Achieve Continuous Operation.
6.35 One Form of Continuous Chromatography Moves the Adsorbent by Rotating The Column.
6.36 Simulated Moving Beds Operate Through A Sequential Switching Scheme to Move the Feed and Product Take-off Points.

Principles of Gradient Elution Chromatography.
7.0 Introduction.
7.1 The System for Carrying Out Gradient Chromatography Is Similar to That For Isocratic Chromatography.
Ion Exchange Gradient Chromatography.
7.2 Linear Gradient Elution In Ion Exchange Chromatography Is Based on Exchange of A Multivalent Protein for a Mono- or Di-valent Salt.
7.3 Separation in Ion Exchange Gradient Chromatography is Driven By the Time-dependent Increase in Salt Concentration in The Mobile Phase.
7.4 Gradient Chromatography Is Often Carried Out In The Middle of a Purification Sequence.
7.5 Purification of Recombinant Proteins From E. coli Requires Steps That First Dissolve and Refold The Proteins.
7.6 Anion Exchange Chromatography Is Prominant In The Purification of Blood Products (Pro- and Anticoagulant Factors).
7.7 Gene Therapy Vectors Can Be Purified by Anion Exchange Chromatography Using Phosphate Buffer and KCl Gradients.
7.8 Process Scale Purification of Plasmid DNA Employs A Sequence of Anion Exchange and Size-exclusion Chromatography.
7.9 An Ion Exchanger is a Solid Material That Carries Exchangeable Cations Or Anions.
7.10 Retention Times and Capacity Factors of Charged Species in Ion Exchange Chromatography are Proportional to Their Charge (with contributions by A. Velayudhan).
7.11 The Definition of the Separation Factor Is Based on Differences of Binding Charges of the Two Components A and B.
7.12 Definitions of Plate Height and Resolution for Linear Gradient Chromatography Are Analogous to Those for Isocratic Chromatography.
7.13 The Plate Height Increases with Increasing Interstitial Velocity in Linear Gradient Elution Chromatography.
7.14 Scale-up of Gradient Chromatography is More Challenging, Than For Isocratic Conditions Since Peak Retention As a Function of Gradient Characteristics Must Either Be Known or Calculated.
7.15 One Approach to Scale-up of Gradient Chromatography Is Based on Maintaining a Constant Gradient Duration.
7.16 Material Balances on Both Modulator and Protein Are Needed to Scale-up Linear Gradient Elution When Column Length And/or Gradient Slope Are Changed.
7.17 Adsorption of The Modulator on The Stationary Phase May Cause Deformation of the Gradient.
7.18 The Concepts of Gradient Chromatography Can Be Extended to Affinity Membranes.
7.19 Electrical Gradients May Also Be Used For Chromatographic Separations.
Summary and Perspectives.

Principles of Bioseparations for Biopharmaceuticals and Recombinant Protein Products.
8.0 Introduction.
8.1 New Biotechnology Products Are the Fastest Growing Area in Bioseparations.
8.2 Bioseparation Processes Have a Significant Impact on Manufacturing Costs.
8.3 Bioseparation Economics Are Secondary to Being First to Market.Until After the New Product is Introduced.
8.4 But Cost Is Important when Success of a New Product Generates Competition.
8.5 Manufacturing Processes for Biologics Are Part of The Product's Regulation.
Insulin Case Study.
8.6 Biosynthetic Human Insulin Is the First Recombinant Polypeptide from E. coli Licensed for Human Use.
8.7 Recovery and Purification of Human Insulin Requires 27 Steps.
8.8 Recovery Process Equipment Volumes are Modest by Chemical Industry Standards.
8.9 Yield Losses are Amplified by the Number of Purification Steps.
8.10 Lys(B28), Pro(B29) Biosynthetic Human Insulin Is a Human Insulin Analog (contributed by Jeffrey C. Baker).
8.11 The Front End Strategy for Manufacture of LysPro Insulin Is Different From Biosynthetic Human Insulin (contributed by Jeffrey Baker).
Tissue Plasminogen Activator.
8.12 Tissue Plasminogen Activator Is the First Recombinant Protein Pharmaceutical from Mammalian Cell Culture for Treatment of Heart Attacks.
8.13 Recombinant Tissue Plasminogen Activator (t-PA) May Be Effective for Treatment of Strokes.
8.14 Tissue Plaminogen Activator Is a Proteolytic Enzyme.
8.15 Recombinant Technology Provides the Only Practical Means of t-PA Production.
8.16 Purification of Tissue Plasminogen Activator Must Remove Cells, Virus, and DNA.
8.17 Limits on Analytical Detection Require Independent Assays for Proving Virus Removal.
8.18 The Presence of u-PA Complicates the Purification of t-PA.
8.19 Process Changes in Manufacture of t-PA, a Biologic, are Subject to Government Regulations.
Classes of Chromatography.
8.20 Purification of Biologics and Drugs are Based on Five Classes of Chromatography.
Ion Exchange Chromatography.
8.21 Ion Exchange is Based on Competition of Charged Species for Stationary Phase Binding Sites.
8.22 Titration Curves Give pK and Guide Selection of Stationary Phase.
8.23 pH, Ion Exchange, Hydrophobic Interactions and Volatility Characteristics of Buffer Components Guide Specification of Buffer Composition.
8.24 Amino Acids Which Flank Charged Residues Can Alter Protein Binding.
8.25 Protein Loading Capacity is Based on Equilibrium Measurements.
8.26 Large Proteins and Small Pore Sizes Decrease Equilibrium Binding Capacity.
8.27 Dynamic (Operational) Capacities are Significantly Lower than Equilibrium Capacities.
8.28 Ion Exchange Chromatography Separates Proteins by Desorption Using Increasing Salt Gradients.
8.29 Amphoteric Properties of Proteins Determine Conditions of Ion Exchange Chromatography.
8.30 Hydroxyapetite (Ca10(PO4)6OH2) Is a Mixed Mode Ion Exchanger With Both Weakly Anionic and Weakly Cationic Functional Groups.
Size Exclusion (Gel Permeation) Chromatography.
8.31 Size Exclusion (Gel Permeation) Separates Proteins Based on Differences in Their Size and Shape.
8.32 Operational Definitions for Elution Volumn in Gel Permeation Are Basic to All Types of Chromatography.
8.33 Representation of Elution Volume on a Chromatogram Depends on Injection Volume of Sample.
8.34 Distribution Coefficients Depend on Molecule Size and Gel Cross-Linking.
8.35 The Distribution Coefficients, Kd, and Kav, Are Not Equivalent.
8.36 Selectivity Curves Plot Measured Values of Kav vs. Logarithm of Molecular Weight.
8.37 Buffer Exchange and Desalting Utilize Gel Permeation.
8.38 Buffer Exchange Can Also be Achieved Using Membranes.
8.39 Gel Permeation Requires a Larger Column Than Ion Exchange Chromatography.
Reversed Phase Chromatography.
8.40 Reversed Phase Chromatography Can Separate Proteins by Solvophobic (Hydrophobic) and Silanophilic (Hydrophilic) Interactions.
8.41 Derivatized Silica Particles Are One Type of Reversed Phase Chromatographic Media.
8.42 Small Particle Silica Columns Benefit From Axial Compression.
8.43 Polymeric Reversed Phase Chromatographic Media Are Based on Polystyrene and Methacrylic Copolymers.
8.44 Experimentally Determined Chromatograms Are the Starting Points for Specifying Solvent Composition for Reversed Phase Chromatography.
8.45 Changes in Relative Peak Retention Occur With Changes in Mobile Phase Polarity.
8.46 Reversed Phase Chromatography Uses Hydrophobic Media and Increasing Gradients of Aqueous Methanol, Acetonitrile or Isopropanol.
8.47 Insulin, a Polypeptide Hormone, is Purified by Reversed Phase, Process Chromatography.
8.48 Purification of Proteins by Reversed Phase Process Chromatography Is not Common.
8.49 Reversed Phase Chromatography is Widely Used for Analysis of Proteins and Peptide Mapping.
8.50 Reversed Phase Process Chromatography of Small Molecules Has a History of Use.
Hydrophobic Interaction.
8.51 Hydrophobic Interaction Chromatography Utilizes Decreasing Aqueous Gradients of Kosmotropes.
8.52 The Type and Concentration of Salt Affects Peak Retention in Hydrophobic Interaction Chromatography.
8.53 Hydrophobic Interaction Chromatography is Suitable for Many Proteins.
Affinity Chromatography.
8.54 Reversible Binding of Enzymes with Immobilized Substrates and Inhibitors Led to Affinity Chromatography.
8.55 Careful Selection of Affinity Stationary Phases Minimizes Non-specific Binding.
8.56 Activation of the Stationary Phase Surface Precedes Covalent Attachment of Affinity Ligands.
8.57 Affinity Chromatography Will Not Be A One Step Purification Processes.
Bioseparations Process Development.
8.58 Downstream Processing Requires Multiple Purification Steps Based on Different Molecular Properties.
8.59 Separation Goals Define Bioseparation Sequences.
8.60 Specification of Separation Goals Facilitates Selection of Separation Methods.
8.61 Separation Strategies Combine Categories, Objectives and Methods Into a Logical Sequence of Purification Steps (Example of SEP-OPS Method).
8.62 Biotechnology Process Development Differs from Chemical Industry Process Development.
8.63 Upstream Production Methods Affect Downstream Purification Strategies.

Affinity Chromatography: Bridge Between Molecular Biology, Combinatorial Methods and Separations Science.
9.0 Introduction.
Affinity Ligands From Combinatorial Libraries.
9.1 Combinatorial Chemistry Creates Libraries of New Peptide Sequences.
9.2 Visual Identification Finds A Needle in The Haystack: One Affinity Ligand Among Thousands of Peptides (Factor IX Example).
9.3 Liquid Chromatography Using 300 mg of Beads Confirms Specificity of Factor IX Binding.
9.4 Cost Effective Synthesis of Kilogram Quantities of An Affinity Peptide Remains A Challenge (Estimating Peptide Cost).
9.5 Combinatorial Synthesis of Carbohydrates and Screening With Biotin Labeled Lectin Gives New Affinity Ligands for Lectins.
9.6 Bovine Serum Albumin (BSA) Suppresses Non-Specific Binding of the Lectin on an Affinity Support.
9.7 The Combination of Combinatorial Chemistry And Computers Will Accelerate Drug Discovery and Catalyst Development.
9.8 Combinatorial Libraries of Small Molecules can be Generated by Solid Phase Synthesis or Solution Based Chemistry.
Receptors, Affinity Ligands, and Acquired Immune Deficiency Syndrome (AIDS).
9.9 HIV-1 Initially Increases Antigens to HIV Core Protein p24 in the Serum of Infected Persons.
9.10 An Immobilized Human Antibody Against the Antigen for the HIV Core Protein, p24, Detects AIDS (ELISA Example).
9.11 HIV is a lentivirus, a type of retrovirus with cytopathic activity.
9.12 Azidothymidine (AZT) Stops HIV Replication by Inhibiting the Enzyme, Reverse Transcriptase.
9.13 Protease Inhibitors Prevent Cleavage of gag- and pol- Encoded Viral Proteins Required For Viral Replication.
9.14 Mutation of HIV to a Drug Resistant Form Is Promoted by Rapid Turnover of a Large Pool of Infected T-Cells.
9.15 New Drug Discovery Is Necessitated By Rapidly Mutating HIV Virus.
9.16 Production Scale-up Is Part of Product Discovery (Case Study of Protease Inhibitors).
9.17 Coreceptors (Affinity Ligands) Facilitate HIV Binding and Infection (CXCR4 and CCR5).
9.18 Receptors Are Important to the Dynamics of HIV Transmission and Pathogenesis.
9.19 A Sensitive and Rapid Screen Helps to Find the CXCR4 (Fusin) Protein Receptor in CD4 Cells.
Phage and Ribosome Display.
9.20 Biocombinatorial Generation of Small Protein Ligands Utilizes Bacterial Viruses (Phages).
9.21 Binding Characteristics of Small Protein Ligands Are Determined by Adding The Phages to Target Molecules Immobilized on Beads.
9.22 Phage Display Technology Has Generated a Protease Inhibitor With High Affinity and Specificity for Plasmin.
9.23 Generation and Screening of Proteins by in vitro Ribosome Display Separates and Amplifies mRNA for a Specific Protein.
Receptor Affinity Chromatography.
9.24 Receptor Affinity Chromatography Selectively Binds Active Forms of Protein.
9.25 Receptor Affinity Chromatography Combines Upstream and Downstream Processing Steps.
9.26 Interleukin-2 Is A Case Study In the Development of Receptor Affinity Chromatography.
9.27 Either

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