Hydrophobic Interaction Chromatography PRINCIPLES AND METHODS c8aeti8n Amersham 18-1020-90 Biosciences Edition AB
Hydrophobic Interaction Chromatography 18-1020-90 PRINCIPLES AND ETHODS M Edition AB
Contents 1.Introduction to 2.Principles of HIC. .11 Theory. .11 HIC vs RPC 12 Factors affecting HIC .13 Type of ligand. 13 Degree of substitution .14 Type of base matrix. .14 Type and concentration of salt .15 Effect of pH. .16 Effect of temperature 17 Additives 18 3.Product Guide 19 BioProcess Media 20 Base matrices .20 Coupling. 21 Chemical stability 21 Physical stability .22 Binding capacity 22 Phenyl Sepharose 6 Fast Flow(low sub)and Phenyl Sepharose 6 Fast Flow (high sub). .23 Butyl Sepharose 4 Fast Flow .24 Phenyl Sepharose High Performance 25 Custom Designed HIC Media 26 HIC Media Test Kit .26
1. Introduction to HIC. 9 2. Principles of HIC. 11 Theory.11 HIC vs RPC .12 Factors affecting HIC.13 Type of ligand .13 Degree of substitution .14 Type of base matrix .14 Type and concentration of salt .15 Effect of pH.16 Effect of temperature .17 Additives .18 3. Product Guide . 19 BioProcess Media .20 Base matrices .20 Coupling .21 Chemical stability .21 Physical stability .22 Binding capacity .22 Phenyl Sepharose 6 Fast Flow (low sub) and Phenyl Sepharose 6 Fast Flow (high sub) .23 Butyl Sepharose 4 Fast Flow .24 Phenyl Sepharose High Performance .25 Custom Designed HIC Media .26 HIC Media Test Kit .26 Contents
Contents Phenyl Sepharose CL-4B and Octyl Sepharose CL-4B 27 Phenyl Superose and Alkyl Superose. .27 4.Experimental Design.29 Hydrophobicity of proteins 29 Multivariate mapping 29 Strategic considerations .30 Choice of HIC media 31 General considerations. 31 Screening experiments 32 Optimizing a HIC step .39 The solute .39 The solvent. .41 Elution. 2 Sample load and flow rate .45 Regeneration .45 Process considerations 46 Method optimization in process chromatography .46 Scaleability .49 Regulatory considerations. 50 5.Experimental Technique.53 Choice of column. 53 Column dimensions. .53 Packing the column .53 Packing Sepharose Fast Flow based HIC gels. .54 Packing Phenyl Sepharose High Performance.55 Packing Sepharose CL-4B based HIC gels .55
Phenyl Sepharose CL-4B and Octyl Sepharose CL-4B .27 Phenyl Superose and Alkyl Superose.27 4. Experimental Design . 29 Hydrophobicity of proteins .29 Multivariate mapping .29 Strategic considerations .30 Choice of HIC media .31 General considerations .31 Screening experiments .32 Optimizing a HIC step .39 The solute .39 The solvent .41 Elution .42 Sample load and flow rate .45 Regeneration .45 Process considerations .46 Method optimization in process . chromatography .46 Scaleability .49 Regulatory considerations.50 5. Experimental Technique . 53 Choice of column .53 Column dimensions.53 Packing the column .53 Packing Sepharose Fast Flow based HIC gels .54 Packing Phenyl Sepharose High Performance .55 Packing Sepharose CL-4B based HIC gels .55 Contents
Contents Use of an adaptor. .55 Checking the packed bed. .56 Prepacked HIC media .58 Sample preparation. .59 Sample composition. .59 Sample volume .59 Sample viscosity .60 Particle content. .60 Sample application. .61 Sample reservoir Sample applicators. .61 Sample loops with valves LV-4 or SRV-4 62 Sample loops or Superloop with valves V-7 or MV-7. .62 Batch separation. Cleaning,sanitization and sterilization procedures .63 Storage of gels and columns 65 Prevention of microbial growth .65 Antimicrobial agents. Storage of unused media. .67 Storage of used media. 67 Storage of packed columns .67 Process considerations. Selecting a column. .68 Aspects of column design 69 Packing large scale columns .71 Scale-up. .74
Use of an adaptor .55 Checking the packed bed .56 Prepacked HIC media .58 Sample preparation .59 Sample composition .59 Sample volume .59 Sample viscosity.60 Particle content.60 Sample application.61 Sample reservoir .61 Sample applicators .61 Sample loops with valves LV-4 or SRV-4 .62 Sample loops or Superloop with valves V-7 or MV-7 .62 Batch separation .63 Cleaning, sanitization and sterilization procedures .63 Storage of gels and columns .65 Prevention of microbial growth .65 Antimicrobial agents .65 Storage of unused media .67 Storage of used media .67 Storage of packed columns .67 Process considerations.68 Selecting a column .68 Aspects of column design .69 Packing large scale columns .71 Scale-up .74 Contents
Contents 6.Applications. .77 Preparative and analytical HIC applications in the research laboratory 77 HIC in combination with ammonium sulphate precipitation. 77 HIC in combination with ion exchange chromatography. .78 HIC in combination with gel filtration. 80 HIC as a"single step"purification technique.81 Analysis of conformational changes with HIC.84 Other HIC application areas in the research laboratory 84 Preparative,large scale applications .85 Purification of a monoclonal antibody for clinical studies of passive immuno- therapy of HIV-1 .85 Purification of recombinant human Epidermal Growth Factor (h-EGF)from yeast.87 Purification of a monoclonal antibody for in vitro diagnostic use 90 Purification of a recombinant Pseudomonas aeruginosa exotoxin,produced in E.Coli. 92 7.References 97 Order from .102
Contents 6. Applications . 77 Preparative and analytical HIC applications in the research laboratory .77 HIC in combination with ammonium sulphate precipitation.77 HIC in combination with ion exchange chromatography .78 HIC in combination with gel filtration .80 HIC as a ”single step” purification technique .81 Analysis of conformational changes with HIC.84 Other HIC application areas in the research laboratory .84 Preparative, large scale applications .85 Purification of a monoclonal antibody for clinical studies of passive immunotherapy of HIV-1 .85 Purification of recombinant human Epidermal Growth Factor (h-EGF) from yeast .87 Purification of a monoclonal antibody for in vitro diagnostic use .90 Purification of a recombinant Pseudomonas aeruginosa exotoxin, produced in E. Coli.92 7. References . 97 Order from. 102