Ion Exchange Chromatography & Chromatofocusing – Ion Exchange Chromatography & Chromatofocusing Principles and Methods Principles and Methods www.gehealthcare.com www.chromatography.amershambiosciences.com 11-0004-21 Edition AA Handbooks from Amersham Biosciences Antibody Purification Handbook Mini Q, Mini S, Mono Q, Mono S, Mono P, RESOURCE, SOURCE, Sepharose, BioProcess, HiTrap, HiLoad, 18-1037-46 HiPrep, Tricorn, FPLC, MiniBeads, MonoBeads, ÄKTA, ÄKTAexplorer, ÄKTApurifier, Sephadex, Sephacel, Sephacryl, BPG, Pharmalyte, ÄKTAprime, ÄKTApilot, STREAMLINE, Hybond, ECL, ECL Plus, Superdex, The Recombinant Protein Handbook PhastGel, PlusOne, PhastSystem, BioDirectory, FineLINE and Drop Design are trademarks of Amersham Protein Amplification and Hydrophobic Interaction Percoll Biosciences Limited. 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Principles and Methods Principles and Methods 18-1157-58 11-0004-21 18-1124-26 Amersham Biosciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden 2-D Electrophoresis Amersham Biosciences UK Limited, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England Affinity Chromatography Microcarrier cell culture using immobilized pH gradients Amersham Biosciences Corp., 800 Centennial Avenue, PO Box 1327, Piscataway NJ 08855, USA Principles and Methods Principles and Methods Principles and Methods Amersham Biosciences Europe GmbH, Munzinger Strasse 9, D-79111 Freiburg, Germany 18-1022-29 18-1140-62 80-6429-60 Amersham Biosciences K.K., Sanken Bldg. 3-25-1, Hyakunincho Amersham Shinjuku-ku, Tokyo 169-0073, Japan Ion Exchange Chromatography & Chromatofocusing Principles and Methods 1 Contents Introduction ............................................................................................................. 7 Symbols ................................................................................................................................................... 8 Common abbreviations .............................................................................................................................. 9 Chapter 1 Principles of ion exchange ..................................................................................... 11 Net surface charge and pH ......................................................................................... 11 Steps in an IEX separation ......................................................................................... 12 Resolution ................................................................................................................ 14 Efficiency ............................................................................................................................................... 15 Selectivity ............................................................................................................................................... 17 Components of ion exchange media ............................................................................ 21 Matrix ..................................................................................................................................................... 21 Functional groups .................................................................................................................................... 23 Binding capacity and recovery .................................................................................... 25 Chapter 2 Ion exchange in practice ........................................................................................ 26 Introduction .............................................................................................................. 26 Media selection ......................................................................................................... 26 Capture .................................................................................................................................................. 26 Intermediate purification .......................................................................................................................... 27 Polishing ................................................................................................................................................ 27 Fast media selection and method development ............................................................. 30 Automated media selection, method development and optimization .............................................................. 30 Manual media selection, method development and optimization ................................................................... 31 Practical considerations for IEX separation ................................................................... 34 pH and ionic strength ............................................................................................................................... 34 Anion or cation exchanger ........................................................................................................................ 35 Strong or weak ion exchangers .................................................................................................................. 36 Buffer selection and preparation ............................................................................................................... 37 Column and media preparation ................................................................................................................. 39 Sample preparation .................................................................................................................................. 39 Sample application .................................................................................................................................. 40 Sample load ............................................................................................................................................ 40 Elution ................................................................................................................................................... 42 Linear gradient elution ............................................................................................................................. 42 Step elution ............................................................................................................................................ 44 pH elution ............................................................................................................................................... 46 Flow rates ............................................................................................................................................... 46 Flow control ............................................................................................................................................ 47 Wash and re-equilibration ......................................................................................................................... 48 Detergents, denaturing agents and other additives ...................................................................................... 48 Analysis of results and further steps ............................................................................ 50 Scaling-up ................................................................................................................ 51 Equipment selection .................................................................................................. 52 Care of ion exchange media ........................................................................................ 52 Troubleshooting ......................................................................................................... 53 BioProcess Media – made for bioprocessing ................................................................. 58 Custom Designed Media ............................................................................................. 58 2 Chapter 3 Ion exchange media ............................................................................................... 59 Introduction .............................................................................................................. 59 MiniBeads: purification or analysis of microgram-milligram quantities with highest resolution ............................................................................................... 60 Purification options .................................................................................................................................. 61 Purification examples ..............................................................................................................................