Chromatography

Edition AC 18-1022-29 Principles and Methods Affinity ChromatographyAntibody Purification Handbook 18-1037-46 The Recombinant Protein Handbook Protein Amplification and Simple Purification 18-1142-75 Protein Purification Handbook 18-1132-29 Ion Exchange Chromatography Principles and Methods 18-1114-21 Affinity Chromatography Principles and Methods 18-1022-29 Hydrophobic Interaction Chromatography Principles and Methods 18-1020-90 Gel Filtration Principles and Methods 18-1022-18 Handbooks from Amersham Pharmacia Biotech Reversed Phase Chromatography Principles and Methods 18-1134-16 Expanded Bed Adsorption Principles and Methods 18-1124-26 Chromatofocusing with Polybuffer and PBE 50-01-022PB Microcarrier cell culture Principles and Methods 18-1140-621 Affinity Chromatography Principles and Methods2 Content Introduction ............................................................................................................. 7 Symbols and abbreviations ......................................................................................................................... 8 Chapter 1 Affinity chromatography in brief ................................................................................ 9 BioProcess Media for large-scale production ................................................................. 12 Custom Designed Media and Columns ......................................................................... 12 Common terms in affinity chromatography ................................................................... 13 Chapter 2 Affinity chromatography in practice ......................................................................... 15 Purification steps ....................................................................................................................................15 Media selection ...................................................................................................................................... 16 Preparation of media and buffers ............................................................................................................... 16 Sample preparation and application ........................................................................................................... 17 Elution .................................................................................................................................................. 18 Flow rates .............................................................................................................................................. 21 Analysis of results and further steps ........................................................................................................... 21 Equipment selection ............................................................................................................................... 21 Troubleshooting ...................................................................................................................................... 22 Chapter 3 Purification of specific groups of molecules ............................................................ 25 Immunoglobulins ....................................................................................................... 25 IgG, IgG fragments and subclasses .............................................................................. 26 HiTrap Protein G HP, Protein G Sepharose 4 Fast Flow, MAbTrap Kit .............................................................28 HiTrap Protein A HP, Protein A Sepharose 4 Fast Flow, HiTrap rProtein A FF, rProtein A Sepharose 4 Fast Flow .............................................................................................................. 33 Monoclonal IgM from hybridoma cell culture ................................................................ 38 HiTrap IgM Purification HP ....................................................................................................................... 38 Avian IgY from egg yolk .............................................................................................. 40 HiTrap IgY Purification HP ........................................................................................................................ 40 Recombinant fusion proteins ...................................................................................... 42 GST fusion proteins ................................................................................................... 42 GST MicroSpin Purification Module, GSTrap FF, Glutathione Sepharose 4 Fast Flow, Glutathione Sepharose 4B ........................................................................................................................ 42 Poly (His) fusion proteins ........................................................................................... 46 His MicroSpin Purification Module, HisTrap Kit, HiTrap Chelating HP, Chelating Sepharose Fast Flow .................................................................................................................. 46 Protein A fusion proteins ............................................................................................ 51 IgG Sepharose 6 Fast Flow ........................................................................................................................ 51 Purification or removal of serine proteases, e.g. thrombin and trypsin, and zymogens ..................................................................... 53 HiTrap Benzamidine FF (high sub), Benzamidine Sepharose 4 Fast Flow (high sub) ........................................533 Serine proteases and zymogens with an affinity for arginine ........................................... 57 Arginine Sepharose 4B ............................................................................................................................. 57 DNA binding proteins ................................................................................................. 59 HiTrap Heparin HP, HiPrep 16/10 Heparin FF, Heparin Sepharose 6 Fast Flow ............................................... 59 Coagulation factors .................................................................................................... 64 HiTrap Heparin HP, HiPrep 16/10 Heparin FF, Heparin Sepharose 6 Fast Flow ............................................... 64 Biotin and biotinylated substances .............................................................................. 65 HiTrap Streptavidin HP, Streptavidin Sepharose High Performance ................................................................ 65 Purification or removal of fibronectin ........................................................................... 68 Gelatin Sepharose 4B ...............................................................................................................................68 Purification or removal of albumin ............................................................................... 69 HiTrap Blue HP, Blue Sepharose 6 Fast Flow ..............................................................................................69 NAD+-dependent dehydrogenases and ATP-dependent kinases ....................................... 72 5' AMP Sepharose 4B, HiTrap Blue HP, Blue Sepharose 6 Fast Flow .............................................................72 NADP+-dependent dehydrogenases and other enzymes with affinity for NADP+ ............... 74 2'5' ADP Sepharose 4B, Red Sepharose CL-6B ...........................................................................................74 Glycoproteins or polysaccharides ................................................................................. 79 Con A Sepharose 4B, Lentil Lectin Sepharose 4B, Agarose Wheat Germ Lectin ............................................... 79 Con A for binding of branched mannoses, carbohydrates with terminal mannose or glucose (aMan > aGlc > GlcNAc) ..........................................................................................................79 Lentil lectin for binding of branched mannoses with fucose linked a(1,6) to the N-acetyl-glucosamine, (aMan > aGlc > GlcNAc) N-acetylglucosamine binding lectins ........................................................................................................................................ 82 Wheat germ lectin for binding of chitobiose core of N-linked oligosaccharides, [GlcNAc(b1,4GlcNAc)1-2 > b GlcNAc] ......................................................................................................... 83 Calmodulin binding proteins: ATPases, adenylate cyclases, protein kinases, phosphodiesterases, neurotransmitters ................................................. 85 Calmodulin Sepharose 4B .........................................................................................................................85 Proteins and peptides with exposed amino acids: His, Cys, Trp, and/or with affinity for metal ions (also known as IMAC, immobilized metal chelate affinity chromatography) ...................................................... 87 HiTrap Chelating HP, Chelating Sepharose Fast Flow, His MicroSpin Purification Module, HisTrap Kit ...........................................................................................87 Thiol-containing substances (purification by covalent chromatography) ........................... 91 Activated Thiol Sepharose 4B, Thiopropyl Sepharose 6B .............................................................................. 91 Chapter 4 Components of an affinity medium ........................................................................... 96 The matrix .............................................................................................................................................. 96 The ligand .............................................................................................................................................. 97 Spacer arms ........................................................................................................................................... 98 Ligand coupling ...................................................................................................................................... 99 Ligand specificity ....................................................................................................................................99Chapter 5 Designing affinity media using pre-activated matrices ............................................ 100 Choosing the matrix ...............................................................................................................................100 Choosing the ligand and spacer arm ......................................................................................................... 100 Choosing the coupling method ................................................................................................................. 100 Coupling the ligand ............................................................................................................................... 102 Binding capacity, ligand density and coupling efficiency ............................................................................103 Binding and elution conditions ................................................................................................................104 Coupling through the primary amine of a ligand .......................................................... 105 HiTrap NHS-activated HP, NHS-activated Sepharose 4 Fast Flow ................................................................105 CNBr-activated Sepharose ....................................................................................................................... 108 Immunoaffinity chromatography .............................................................................................................. 112 Coupling small ligands through amino or carboxyl groups via a spacer arm ...................................................................................................... 113 EAH Sepharose 4B and ECH Sepharose 4B ..............................................................................................113 Coupling through hydroxy, amino or thiol groups via a 12-carbon spacer arm ...................................................................................... 116 Epoxy-activated Sepharose 6B ................................................................................................................. 116 Coupling through a thiol group .................................................................................. 120 Thiopropyl Sepharose 6B ........................................................................................................................ 120 Coupling other functional groups ............................................................................... 121 Chapter 6 Affinity chromatography and CIPP ........................................................................... 123 Applying CIPP .......................................................................................................... 124 Selection and combination of purification techniques .................................................. 124 Appendix 1 .......................................................................................................... 129 Sample preparation ................................................................................................. 129 Sample stability ....................................................................................................................................129 Sample clarification ...............................................................................................................................130 Specific sample preparation steps ............................................................................. 131 Resolubilization of protein precipitates ..................................................................................................... 133 Buffer exchange and desalting .................................................................................. 134 Removal of lipoproteins ............................................................................................ 137 Removal of phenol red ............................................................................................. 137 Removal of low molecular weight contaminants .......................................................... 137 Appendix 2 .......................................................................................................... 139 Selection of purification equipment ........................................................................... 139 Appendix 3 .......................................................................................................... 140 Column packing and preparation ............................................................................... 140Appendix 4 .......................................................................................................... 142 Converting from linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa ............................................................................. 142 Appendix 5 .......................................................................................................... 143 Conversion data: proteins, column pressures .............................................................. 143 Column pressures ................................................................................................................................. 143 Appendix 6 .......................................................................................................... 144 Table of amino acids ................................................................................................ 144 Appendix 7 .......................................................................................................... 146 Kinetics in affinity chromatography ........................................................................... 146 Appendix 8 .......................................................................................................... 151 Analytical assays during purification .......................................................................... 151 Appendix 9 .......................................................................................................... 153 Storage of biological samples .................................................................................... 153 Additional reading and reference material ............................................................. 154 Ordering information ............................................................................................ 1557 Introduction Biomolecules are purified using purification techniques that separate according to differences in specific properties, as shown in Figure 1. Property Technique* Biorecognition (ligand specificity) Affinity chromatography Charge Ion exchange chromatography Size Gel filtration (sometimes called size exclusion) Hydrophobicity Hydrophobic interaction chromatography Reversed phase chromatography *Expanded bed adsorption is a technique used for large-scale purification. Proteins can be purified from crude sample without the need for separate clarification, concentration and initial purification to remove particulate matter. The STREAMLINE™ adsorbents, used for expanded bed adsorption, capture the target molecules using the same principles as affinity, ion exchange or hydrophobic interaction chromatography. Fig. 1. Separation principles in chromatographic purification. Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatographic matrix. The technique offers high selectivity, hence high resolution, and usually high capacity for the protein(s) of interest. Purification can be in the order of several thousand-fold and recoveries of active material are generally very high. Affinity chromatography is unique in purification technology since it is the only technique that enables the purification of a biomolecule on the basis of its biological function or individual chemical structure. Purification that would otherwise be time-consuming, difficult or even impossible using other techniques can often be easily achieved with affinity chromatography. The technique can be used to separate active biomolecules from denatured or functionally different forms, to isolate pure substances present at low concentration in large volumes of crude sample and also to remove specific contaminants. Amersham Pharmacia Biotech offers a wide variety of prepacked columns, ready to use media, and pre-activated media for ligand coupling. Gel filtration Hydrophobic interaction Ion exchange Affinity Reversed phase8 This handbook describes the role of affinity chromatography in the purification of biomolecules, the principle of the technique, the media available and how to select them, application examples and detailed instructions for the most commonly performed procedures. Practical information is given as a guide towards obtaining the best results. The illustration on the inside cover shows the range of handbooks that have been produced by Amersham Pharmacia Biotech to ensure that purification with any chromatographic technique becomes a simple and efficient procedure at any scale and in any laboratory. Symbols and abbreviations this symbol indicates general advice which can improve procedures or provide recommendations for action under specific situations. this symbol denotes advice which should be regarded as mandatory and gives a warning when special care should be taken. this symbol highlights troubleshooting advice to help analyse and resolve difficulties that may occur. chemicals, buffers and equipment. experimental protocol. PBS phosphate buffered saline (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4).9 Chapter 1 Affinity chromatography in brief Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatographic matrix. The technique is ideal for a capture or intermediate step in a purification protocol and can be used whenever a suitable ligand is available for the protein(s) of interest. With high selectivity, hence high resolution, and high capacity for the protein(s) of interest, purification levels in the order of several thousand-fold with high recovery of active material are achievable. Target protein(s) is collected in a purified, concentrated form. Biological interactions between ligand and target molecule can be a result of electrostatic or hydrophobic interactions, van der Waals' forces and/or hydrogen bonding. To elute the target molecule from the affinity medium the interaction can be reversed, either specifically using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity. In a single step, affinity purification can offer immense time-saving over less selective multistep procedures. The concentrating effect enables large volumes to be processed. Target molecules can be purified from complex biological mixtures, native forms can be separated from denatured forms of the same substance and small amounts of biological material can be purified from high levels of contaminating substances. For an even higher degree of purity, or when there is no suitable ligand for affinity purification, an efficient multi-step process must be developed using the purification strategy of Capture, Intermediate Purification and Polishing (CIPP). When applying this strategy affinity chromatography offers an ideal capture or intermediate step in any purification protocol and can be used whenever a suitable ligand is available for the protein of interest. Successful affinity purification requires a biospecific ligand that can be covalently attached to a chromatographic matrix. The coupled ligand must retain its specific binding affinity for the target molecules and, after washing away unbound material, the binding between the ligand and target molecule must be reversible to allow the target molecules to be removed in an active form. Any component can be used as a ligand to purify its respective binding partner. Some typical biological interactions, frequently used in affinity chromatography, are listed below: • Enzyme ! substrate analogue, inhibitor, cofactor. • Antibody ! antigen, virus, cell. • Lectin ! polysaccharide, glycoprotein, cell surface receptor, cell. • Nucleic acid ! complementary base sequence, histones, nucleic acid polymerase, nucleic acid binding protein. • Hormone, vitamin ! receptor, carrier protein. • Glutathione ! glutathione-S-transferase or GST fusion proteins. • Metal ions ! Poly (His) fusion proteins, native proteins with histidine, cysteine and/or tryptophan residues on their surfaces.10 Column Volumes (cv) begin sample application change to elution buffer x cv 1-2 cv >1 1-2 cv cv 1-2 cv equilibration adsorption of sample and elution of unbound material wash away unbound material elute bound protein(s) Absorbance re-equilibration Fig. 2. Typical affinity purification. 4. Affinity medium is re-equilibrated with binding buffer. 3. Target protein is recovered by changing conditions to favour elution of the bound molecules. Elution is performed specifically, using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity.Target protein is collected in a purified, concentrated form. 2. Sample is applied under conditions that favour specific binding of the target molecule(s) to a complementary binding substance (the ligand). Target substances bind specifically, but reversibly, to the ligand and unbound material washes through the column. 1. Affinity medium is equilibrated in binding buffer. Affinity chromatography is also used to remove specific contaminants, for example Benzamidine Sepharose™ 6 Fast Flow can remove serine proteases, such as thrombin and Factor Xa. Figure 2 shows the key stages in an affinity purification.11 The high selectivity of affinity chromatography enables many separations to be achieved in one simple step, including, for example, common operations such as the purification of monoclonal antibodies or fusion proteins. A wide variety of prepacked columns, ready to use media, and pre-activated media for ligand coupling through different functional groups, makes affinity chromatography readily available for a broad range of applications. To save time, the HiTrap™ column range (Table 1) is excellent for routine laboratory scale applications in which the risk of cross-contamination between samples must be eliminated, for purification from crude samples or for fast method development before scaling up purification. HiTrap columns can be operated with a syringe, a peristaltic pump or any ÄKTA™design chromatography system. Several HiTrap columns can be connected in series to increase purification capacity and all columns are supplied with detailed protocols for use. Table 1. HiTrap and HiPrep™ affinity columns for laboratory scale purification. Application HiTrap and HiPrep columns Isolation of human immunoglobulins IgG, fragments and subclasses HiTrap rProtein A FF, 1 ml and 5 ml IgG, fragments and subclasses HiTrap Protein A HP, 1 ml and 5 ml IgG, fragments and subclasses including human IgG3 HiTrap Protein G HP, 1 ml and 5 ml strong affinity for monoclonal mouse IgG1 and rat IgG MAbTrap™ Kit Avian IgY from egg yolk HiTrap IgY Purification HP, 5 ml Mouse and human IgM HiTrap IgM Purification HP, 1 ml Purification of fusion proteins (His)6 fusion proteins HisTrap™ Kit HiTrap Chelating HP, 1 ml and 5 ml GST fusion proteins GSTrap™ FF, 1 ml and 5 ml Other Group Specific Media Albumin and nucleotide-requiring enzymes HiTrap Blue HP, 1 ml and 5 ml Proteins and peptides with exposed His, Cys or Trp HiTrap Chelating HP, 1 ml and 5 ml Biotinylated substances HiTrap Streptavidin HP, 1 ml DNA binding proteins and coagulation factors HiTrap Heparin HP, 1 ml and 5 ml HiPrep 16/10 Heparin FF, 20 ml Trypsin-like serine proteases including Factor Xa, thrombin and trypsin HiTrap Benzamidine FF (high sub), 1 ml and 5 ml Matrix for preparation of affinity media. Coupling via primary amines HiTrap NHS-activated HP, 1 ml and 5 ml12 BioProcess Media for large-scale production Specific BioProcess™ Media have been designed for each chromatographic stage in a process from Capture to Polishing. Large capacity production integrated with clear ordering and delivery routines ensure that BioProcess Media are available in the right quantity, at the right place, at the right time. Amersham Pharmacia Biotech can assure future supplies of BioProcess Media, making them a safe investment for long-term production. The media are produced following validated methods and tested under strict control to fulfil high performance specifications. A certificate of analysis is available with each order. Regulatory Support Files contain details of performance, stability, extractable compounds and analytical methods. The essential information in these files gives an invaluable starting point for process validation, as well as providing support for submissions to regulatory authorities. Using BioProcess Media for every stage results in an easily validated process. High flow rate, high capacity and high recovery contribute to the overall economy of an industrial process. All BioProcess Media have chemical stability to allow efficient cleaning and sanitization procedures. Packing methods are established for a wide range of scales and compatible large-scale columns and equipment are available. Please refer to the latest BioProcess Products Catalogue from Amersham Pharmacia Biotech for further details of our products and services for large-scale production. Custom Designed Media and Columns Prepacked columns, made according to the client's choice from the Amersham Pharmacia Biotech range of columns and media, can be supplied by the Custom Products Group. Custom Designed Media (CDM) can be produced for specific industrial process separations when suitable media are not available from the standard range. The CDM group at Amersham Pharmacia Biotech works in close collaboration with the user to design, manufacture, test and deliver media for specialized separation requirements. When a chromatographic step is developed to be an integral part of a manufacturing process, the choice of column is important to ensure consistent performance and reliable operation. Amersham Pharmacia Biotech provides a wide range of columns that ensures the highest performance from all our purification media and meets the demands of modern pharmaceutical manufacturing. Please ask your local representative for further details of CDM products and services.13 Matrix: for ligand attachment. Matrix should be chemically and physically inert. Spacer arm: used to improve binding between ligand and target molecule by overcoming any effects of steric hindrance. Ligand: molecule that binds reversibly to a specific target molecule or group of target molecules. Binding: buffer conditions are optimized to ensure that the target molecules interact effectively with the ligand and are retained by the affinity medium as all other molecules wash through the column. Elution: buffer conditions are changed to reverse (weaken) the interaction between the target molecules and the ligand so that the target molecules can be eluted from the column. Wash: buffer conditions that wash unbound substances from the column without eluting the target molecules or that re-equilibrate the column back to the starting conditions (in most cases the binding buffer is used as a wash buffer). Ligand coupling: covalent attachment of a ligand to a suitable pre-activated matrix to create an affinity medium. Pre-activated matrices: matrices which have been chemically modified to facilitate the coupling of specific types of ligand. Common terms in affinity chromatography1415 Chapter 2 Affinity chromatography in practice This chapter provides guidance and advice that is generally applicable to any affinity purification. The first step towards a successful purification is to determine the availability of a suitable ligand that interacts reversibly with the target molecule or group of molecules. Ready to use affinity media, often supplied with complete separation protocols, already exist for many applications. The contents section of this handbook lists the full range of affinity media from Amersham Pharmacia Biotech according to the specific molecule or group of molecules to be purified. Application-and product-specific information and advice for these media are supplied in other sections of this handbook. Practical information specific to the use of pre-activated matrices for the preparation of affinity medium is covered in Chapter 5. Purification steps 4. Affinity medium is re-equilibrated with binding buffer. 3. Target protein is recovered by changing conditions to favour elution of the bound molecules. Elution is performed specifically, using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity. Target protein is collected in a purified, concentrated form. 2. Sample is applied under conditions that favour specific binding of the target molecule(s) to a complementary binding substance (the ligand). Target substances bind specifically, but reversibly, to the ligand and unbound material washes through the column. 1. Affinity medium is equilibrated in binding buffer. Column Volumes (cv) begin sample application change to elution buffer x cv 1-2 cv >1 1-2 cv cv 1-2 cv equilibration adsorption of sample and elution of unbound material wash away unbound material elute bound protein(s) Absorbance re-equilibration Fig. 3. Typical affinity purification.16 Figure 4 shows the simple procedure used to perform affinity purification on prepacked HiTrap columns. Equilibrate column with binding buffer Apply sample Wash with binding buffer Waste Collect Elute with elution buffer Collect fractions 3 min 5-15 min 2 min Fig. 4. HiTrap columns may be used with a syringe, a peristaltic pump or a liquid chromatography system (see Selection of Purification Equipment, Appendix 2) and are supplied with a detailed protocol to ensure optimum results. Media selection A ligand already coupled to a matrix is the simplest solution. Selecting prepacked columns such as HiTrap or HiPrep will not only be more convenient, but will also save time in method optimization as these columns are supplied with detailed instructions for optimum performance. If a ligand is available, but needs to be coupled to a pre-activated matrix, refer to Chapter 5. If no suitable ligand is available, decide whether it is worth the time and effort involved to obtain a ligand and to develop a specific affinity medium. In many cases, it may be more convenient to use alternative purification techniques such as ion exchange or hydrophobic interaction chromatography. Preparation of media and buffers Storage solutions and preservatives should be washed away thoroughly before using any affinity medium. Re-swell affinity media supplied as freeze-dried powders in the correct buffer as recommended by the manufacturer. Use high quality water and chemicals. Solutions should be filtered through 0.45 μm or 0.22 μm filters. Reuse of affinity media depends on the nature of the sample and should only be performed with identical samples to prevent cross-contamination. If an affinity medium is to be used routinely, care must be taken to ensure that any contaminants from the crude sample can be removed by procedures that do not damage the ligand. Binding and elution buffers are specific for each affinity medium since it is their influence on the interaction between the target molecule and the ligand that facilitates the affinitybased separation. Some affinity media may also require a specific buffer in order to make the medium ready for use again.17 Avoid using magnetic stirrers as they may damage the matrix. Use mild rotation or end-over-end stirring. Sample preparation and application Samples should be clear and free from particulate matter. Simple steps to clarify a sample before beginning purification will avoid clogging the column, may reduce the need for stringent washing procedures and can extend the life of the chromatographic medium. Appendix 1 contains an overview of sample preparation techniques. If possible, test the affinity of the ligand: target molecule interaction. Too low affinity will result in poor yields since the target protein may wash through or leak from the column during sample application. Too high affinity will result in low yields since the target molecule may not dissociate from the ligand during elution. Binding of the target protein may be made more efficient by adjusting the sample to the composition and pH of the binding buffer: perform a buffer exchange using a desalting column or dilute in binding buffer (see page 134). Sample preparation techniques should ensure that components known to interfere with binding (the interaction between the target molecule and the ligand) are removed. Since affinity chromatography is a binding technique, the sample volume does not affect the separation as long as conditions are chosen to ensure that the target protein binds strongly to the ligand. It may be necessary to test for a flow rate that gives the most efficient binding during sample application since this parameter can vary according to the specific interaction between the target protein and the ligand and their concentrations. The column must be pre-equilibrated in binding buffer before beginning sample application. For interactions with strong affinity between the ligand and the target molecule that quickly reach equilibrium, samples can be applied at a high flow rate. However, for interactions with weak affinity and/or slow equilibrium, a lower flow rate should be used. The optimal flow rate to achieve efficient binding may vary according to the specific interaction and should be determined when necessary. Further details on the kinetics involved in binding and elution from affinity media are covered in Appendix 7. When working with very weak affinity interactions that are slow to reach equilibrium, it may be useful to stop the flow after applying the sample to allow more time for the interaction to take place before continuing to wash the column. In some cases, applying the sample in aliquots may be beneficial. Do not begin elution of target substances until all unbound material has been washed through the column by the binding buffer (determined by UV absorbance at 280 nm). This will improve the purity of the eluted target substance.18 Elution There is no generally applicable elution scheme for all affinity media. Reference to manufacturer's instructions, the scientific literature and a few simple rules should result in an effective elution method that elutes the target protein in a concentrated form. Elution methods may be either selective or non-selective, as shown in Figure 5. Method 1 The simplest case. A change of buffer composition elutes the bound substance without harming either it or the ligand. Method 2 Extremes of pH or high concentrations of chaotropic agents are required for elution, but these may cause permanent or temporary damage. Methods 3 and 4 Specific elution by addition of a substance that competes for binding. These methods can enhance the specificity of media that use group-specific ligands. Fig. 5. Elution methods. When substances are very tightly bound to the affinity medium, it may be useful to stop the flow for some time after applying eluent (10 min. to 2 h is commonly used) before continuing elution. This gives more time for dissociation to take place and thus helps to improve recoveries of bound substances. Selective elution methods are applied in combination with group-specific ligands whereas non-selective elution methods are used in combination with highly specific ligands. Forces that maintain the complex include electrostatic interactions, hydrophobic effects and hydrogen bonding. Agents that weaken these interactions may be expected to function as efficient eluting agents. The optimal flow rate to achieve efficient elution may vary according to the specific interaction and should be determined when necessary. Further details on the kinetics involved in binding and elution of target molecules from affinity media are covered in Appendix 7. A compromise may have to be made between the harshness of the eluent required for elution and the risk of denaturing the eluted material or damaging the ligand on the affinity medium. Ready to use affinity media from Amersham Pharmacia Biotech are supplied with recommendations for the most suitable elution buffer to reverse the interaction between the ligand and target protein of the specific interaction. Each of these recommendations will be based on one of the following elution methods:19 pH elution A change in pH alters the degree of ionization of charged groups on the ligand and/or the bound protein. This change may affect the binding sites directly, reducing their affinity, or cause indirect changes in affinity by alterations in conformation. A step decrease in pH is the most common way to elute bound substances. The chemical stability of the matrix, ligand and target protein determines the limit of pH that may be used. If low pH must be used, collect fractions into neutralization buffer such as 1 M Tris-HCl, pH 9 (60–200 μl per ml eluted fraction) to return the fraction to a neutral pH. The column should also be re-equilibrated to neutral pH immediately. Ionic strength elution The exact mechanism for elution by changes in ionic strength will depend upon the specific interaction between the ligand and target protein. This is a mild elution using a buffer with increased ionic strength (usually NaCl), applied as a linear gradient or in steps. Enzymes usually elute at a concentration of 1 M NaCl or less. Competitive elution Selective eluents are often used to separate substances on a group specific medium or when the binding affinity of the ligand/target protein interaction is relatively high. The eluting agent competes either for binding to the target protein or for binding to the ligand. Substances may be eluted either by a concentration gradient of a single eluent or by pulse elution, see page 22. When working with competitive elution the concentration of competing compound should be similar to the concentration of the coupled ligand. However, if the free competing compound binds more weakly than the ligand to the target molecule, use a concentration ten-fold higher than that of the ligand. Reduced polarity of eluent Conditions are used to lower the polarity of the eluent promote elution without inactivating the eluted substances. Dioxane (up to 10%) or ethylene glycol (up to 50%) are typical of this type of eluent. Chaotropic eluents If other elution methods fail, deforming buffers, which alter the structure of proteins, can be used, e.g. chaotropic agents such as guanidine hydrochloride or urea. Chaotropes should be avoided whenever possible since they are likely to denature the eluted protein.20 0 0.1 0.2 00.1 0.2 0.3 0.4 A 280 nm 0.5 0 45 65 min Imidazole (M) 0.3 UV 280 nm Programmed elution buffer conc. (His) fusion protein 6 1 2 1: selected imidazole concentration for elution of impurities 2: selected imidazole concentration for elution of pure (His) 6 fusion protein Gradient and step elution Figure 6 shows examples of step and gradient elution conditions. For prepacked affinity HiTrap columns, supplied with predefined elution conditions, a step elution using a simple syringe can be used. HiTrap columns can also be used with a chromatography system such as ÄKTAprime. The use of a chromatography system is essential when gradient elution is required. Sample: Clarified homogenate of E. coli expressing His fusion protein Column: HiTrap Chelating HP 1 ml column charged with Ni2+ Binding buffer: 20 mM sodium phosphate, 0.5 M sodium chloride, 10 mM imidazole, pH 7.4 Elution buffer: 20 mM sodium phosphate, 0.5 M sodium chloride, 0.5 M imidazole, pH 7.4 Flow: 1 ml/min System: ÄKTAprime Fig. 6a. Step elution. A280 Time/vol. Binding conditions Elution conditions A280 Time/vol. Binding conditions Linear change in elution conditions Fig. 6b. Gradient elution. During development and optimization of affinity purification, use a gradient elution to scan for the optimal binding or elution conditions, as shown in Figure 7 and Figure 8. Fig. 7. Gradient elution of a (His)6 fusion protein.21 Fig. 8. Scouting for optimal elution pH of a monoclonal IgG1 from HiTrap rProtein A FF, using a pH gradient. Flow rates It is not possible to specify a single optimal flow rate in affinity chromatography because dissociation rates of ligand/target molecule interactions vary widely. For ready to use affinity media follow the manufacturer's instructions and optimize further if required: -determine the optimal flow rate to achieve efficient binding -determine the optimal flow rate for elution to maximize recovery -determine the maximum flow rate for column re-equilibration to minimize total run times To obtain sharp elution curves and maximum recovery with minimum dilution of separated molecules, use the lowest acceptable flow rate. Analysis of results and further steps The analysis of results from the first separation can indicate if the purification needs to be improved to increase the yield, achieve higher purity, speed up the separation or increase the amount of sample that can be processed in a single run. Commonly used assays are outlined in Appendix 8. It is generally recommended to follow any affinity step with a second technique, such as a high resolution gel filtration to remove any aggregates, or ligands that may have leached from the medium. For example, Superdex™ can be used to separate molecules, according to differences in size, and to transfer the sample into storage buffer, removing excess salt and other small molecules. The chromatogram will also give an indication of the homogeneity of the purified sample. Alternatively, a desalting column that gives low resolution, but high sample capacity, can be used to quickly transfer the sample into storage buffer and remove excess salt (see page 134). Equipment selection Appendix 2 provides a guide to the selection of purification systems. A 280 nm 3.0 4.0 5.0 6.0 7.0 150 200 250 0 0.2 0.4 0.6 mlpH A pH selected for elution in a step gradient pH 280 Sample: Cell culture supernatant containing monoclonal IgG1, 90 ml Column: HiTrap rProtein A FF, 1 ml Binding buffer: 100 mM sodium phosphate, 100 mM sodium citrate, 2.5 M sodium chloride, pH 7.4 Elution buffer: 100 mM sodium phosphate, 100 mM sodium citrate, pH-gradient from 7.4 to 3.0 Flow: 1 ml/min System: ÄKTAFPLC™22 Troubleshooting This section focuses on practical problems that may occur when running a chromatography column. The diagrams below give an indication of how a chromatogram may deviate from the ideal during affinity purification and what measures can be taken to improve the results. Target elutes as a sharp peak. Satisfactory result • If it is difficult or impossible to retain biological activity when achieving this result, either new elution conditions or a new ligand must be found. • If using low pH for elution, collect the fractions in neutralization buffer (60–200 μl 1 M Tris-HCl, pH 9.0 per ml eluted fraction). Target is a broad, low peak that elutes while binding buffer is being applied • Find better binding conditions. A280 ml Binding buffer Elution buffer Eluted target Flow through (unbound material) A280 ml Binding buffer Eluted target Flow through (unbound material) A280 ml Binding buffer Elution buffer Eluted target Flow through (unbound material) A280 ml Binding buffer Elution buffer Eluted target Flow through (unbound material) A280 ml Binding buffer Elution buffer Eluted target Wait Flow through (unbound material) A280 ml Binding buffer Elution buffer Eluted target Flow through (unbound material) Target elutes in a broad, low peak • Try different elution conditions. • If using competitive elution, increase the concentration of the competitor in the elution buffer. • Stop flow intermittently during elution to allow time for the target molecule to elute and so collect the target protein in pulses (see second figure beneath). Note: This result may also be seen if the target protein has denatured and aggregated on the column or if there is non-specific binding. Some of the target molecule elutes as a broad, low peak while still under binding conditions • Allow time for the sample to bind and/or apply sample in aliquots, stopping the flow for a few minutes between each sample application (see second figure beneath).23 Situation Cause Remedy Protein does not bind Sample has not been filtered properly. Clean the column, filter the sample or elute as expected. and repeat. Sample has altered during storage. Prepare fresh samples. Sample has wrong pH or buffer Use a desalting column to transfer sample into conditions are incorrect. the correct buffer (see page 134). Solutions have wrong pH. Calibrate pH meter, prepare new solutions and try again. The column is not equilibrated Repeat or prolong the equilibration step. sufficiently in the buffer. Proteins or lipids have Clean and regenerate the column or use a precipitated on the column. new column. Column is overloaded with sample. Decrease the sample load. Microbial growth has occurred Microbial growth rarely occurs in columns in the column. during use, but, to prevent infection of packed columns, store in 20% ethanol when possible. Precipitation of protein in the Clean the column, exchange or clean the filter column filter and/or at the top or use a new column. of the bed. Low recovery of activity, but Protein may be unstable or Determine the pH and salt stability of the normal recovery of protein. inactive in the elution buffer. protein. Collect fractions into neutralization buffer such as 1 M Tris-HCl, pH 9 (60–200 μl per fraction). Enzyme separated from Test by pooling aliquots from the fractions and co-factor or similar. repeating the assay. Lower yield than expected. Protein may have been Add protease inhibitors to the sample and degraded by proteases. buffers to prevent proteolytic digestion. Run sample through a medium such as Benzamidine 4 Fast Flow (high sub) to remove serine proteases. Adsorption to filter during Use another type of filter. sample preparation. Sample precipitates. May be caused by removal of salts or unsuitable buffer conditions. Hydrophobic proteins. Use chaotropic agents, polarity reducing agents Protein is still attached to ligand. or detergents. More activity is Different assay conditions have Use the same assay conditions for all the recovered than was been used before and after assays in the purification scheme. applied to the column. the chromatographic step. Removal of inhibitors during separation. Reduced or poor flow Presence of lipoproteins or Remove lipoproteins and aggregrates during through the column. protein aggregates. sample preparation (see Appendix 1). Protein precipitation in the Modify the eluent to maintain stability. column caused by removal of stabilizing agents during fractionation. Clogged column filter. Replace the filter or use a new column. Always filter samples and buffer before use. Clogged end-piece or Remove and clean or use a new column. adaptor or tubing. Precipitated proteins. Clean the column using recommended methods or use a new column. Bed compressed. Repack the column, if possible, or use a new column. Microbial growth. Microbial growth rarely occurs in columns during use, but, to prevent infection of packed columns, store in 20% ethanol when possible.24 Situation Cause Remedy Back pressure increases Turbid sample. Improve sample preparation (see Appendix 1). during a run or during Improve sample solubility by the addition of successive runs. ethylene glycol, detergents or organic solvents. Precipitation of protein in the column Clean using recommended methods. Exchange filter and/or at the top of the bed. or clean filter or use a new column. Include any additives that were used for initial sample solubilization in the solutions used for chromatography. Bubbles in the bed. Column packed or stored at Remove small bubbles by passing de-gassed cool temperature and then buffer upwards through the column. Take warmed up. special care if buffers are used after storage in a fridge or cold-room. Do not allow column to warm up due to sunshine or heating system. Repack column, if possible, (see Appendix 3). Buffers not properly de-gassed. De-gas buffers thoroughly. Cracks in the bed. Large air leak in column. Check all connections for leaks. Repack the column if possible (see Appendix 3). Distorted bands as sample Air bubble at the top of the Re-install the adaptor taking care to avoid air runs into the bed. column or in the inlet adaptor. bubbles. Particles in buffer or sample. Filter or centrifuge the sample. Protect buffers from dust. Clogged or damaged net in Dismantle the adaptor, clean or replace the net. upper adaptor. Keep particles out of samples and eluents. Distorted bands as Column poorly packed. Suspension too thick or too thin. Bed packed at sample passes down a temperature different from run. the bed. Bed insufficiently packed (too low packing pressure, too short equilibration). Column packed at too high pressure.25 Chapter 3 Purification of specific groups of molecules A group specific medium has an affinity for a group of related substances rather than for a single type of molecule. The same general ligand can be used to purify several substances (for example members of a class of enzymes) without the need to prepare a new medium for each different substance in the group. Within each group there is either structural or functional similarity. The specificity of the affinity medium derives from the selectivity of the ligand and the use of selective elution conditions. Immunoglobulins The diversity of antibody-antigen interactions has created many uses for antibodies and antibody fragments. They are used for therapeutic and diagnostic applications as well as for immunochemical techniques within general research. The use of recombinant technology has greatly expanded our ability to manipulate the characteristics of these molecules to our advantage. The potential exists to create an infinite number of combinations between immunoglobulins and immunoglobulin fragments with tags and other selected proteins. A significant advantage for the purification of antibodies and their fragments is that a great deal of information is available about the properties of the target molecule and the major contaminants, no matter whether the molecule is in its a native state or has been genetically engineered and no matter what the source material. The Antibody Purification Handbook from Amersham Pharmacia Biotech presents the most effective and frequently used strategies for sample preparation and purification of the many different forms of antibodies and antibody fragments used in the laboratory. The handbook also includes more detailed information on antibody structure and classification, illustrated briefly here in Figures 9 and 10. Fig. 9. H2L2 structure of a typical immunoglobulin.26 Fig. 10. Antibody classes. IgG, IgG fragments and subclasses The basis for purification of IgG, IgG fragments and subclasses is the high affinity of protein A and protein G for the Fc region of polyclonal and monoclonal IgG-type antibodies, see Figure 9. Protein A and protein G are bacterial proteins (from Staphylococcus aureus and Streptococcus, respectively) which, when coupled to Sepharose, create extremely useful, easy to use media for many routine applications. Examples include the purification of monoclonal IgG-type antibodies, purification of polyclonal IgG subclasses, and the adsorption and purification of immune complexes involving IgG. IgG subclasses can be isolated from ascites fluid, cell culture supernatants and serum. Table 2 shows a comparison of the relative binding strengths of protein A and protein G to different immunoglobulins compiled from various publications. A useful reference on this subject is also: Structure of the IgG-binding regions of streptococcal Protein G, EMBO J., 5, 1567–1575 (1986). Binding strengths are tested with free protein A or protein G and can be used as a guide to predict the binding behaviour to a protein A or protein G purification medium. However, when coupled to an affinity matrix, the interaction may be altered. For example, rat IgG1 does not bind to protein A, but does bind to Protein A Sepharose. Antibody classes Characteristic IgG k or l k or l k or l k or l k or l g Y structure Light chain Heavy chain IgM IgA IgE IgD m a e d27 Table 2. Relative binding strengths of protein A and protein G to various immunoglobulins. No binding: -, relative strength of binding: +, ++, +++, ++++. Protein A Protein G Species Subclass binding binding Human IgA variable -IgD --IgE IgG1 ++++ ++++ IgG2 ++++ ++++ IgG3 -++++ IgG4 ++++ ++++ IgMA variable -Chicken IgY --Avian egg yolk IgYB --Cow ++ ++++ Dog ++ + Goat -++ Guinea pig IgG1 ++++ ++ IgG2 ++++ ++ Hamster + ++ Horse ++ ++++ Koala -+ Llama -+ Monkey (rhesus) ++++ ++++ Mouse IgG1 + ++++ IgG2a ++++ ++++ IgG2b +++ +++ IgG3 ++ +++ IgMA variable -Pig +++ +++ Rabbit no distinction ++++ +++ Rat IgG1 -+ IgG2a -++++ IgG2b -++ IgG3 + ++ Sheep +/-++ A Purify using HiTrap IgM Purification HP columns. B Purify using HiTrap IgY Purification HP columns. Single step purification based on Fc region specificity will co-purify host IgG and may even bind trace amounts of serum proteins. For any preparation that must be free of even trace amounts of contaminating IgG, immunospecific affinity using anti-host IgG antibodies as the ligand to remove host IgG or using target specific antigen to avoid binding host IgG, ion exchange and/or hydrophobic interaction chromatography may be better alternatives (see Chapter 6). Both protein A and a recombinant protein A are available, with similar specificities for the Fc region of IgG. The recombinant protein A has been engineered to include a C-terminal cysteine that enables a single-point coupling to Sepharose. Single point coupling often results in an enhanced binding capacity. Genetically engineered antibodies and antibody fragments can have altered biological properties and also altered properties to facilitate their purification. For example, tags can be introduced into target molecules for which no affinity media were previously available thus creating a fusion protein that can be effectively purified by affinity chromatography. Details for the purification of tagged proteins are covered in the section Recombinant Fusion Proteins on page 42 of this handbook. For information on the purification of28 recombinant proteins in general, refer to The Recombinant Protein Handbook: Protein Amplification and Simple Purification from Amersham Pharmacia Biotech. HiTrap Protein G HP, Protein G Sepharose 4 Fast Flow, MAbTrap Kit Protein G, a cell surface protein from Group G streptococci, is a type III Fc-receptor. Protein G binds through a non-immune mechanism. Like protein A, it binds specifically to the Fc region of IgG, but it binds more strongly to several polyclonal IgGs (Table 2) and to human IgG3. Under standard buffer conditions, protein G binds to all human subclasses and all mouse IgG subclasses, including mouse IgG1. Protein G also binds rat IgG2a and IgG2b, which either do not bind or bind weakly to protein A. Amersham Pharmacia Biotech offers a recombinant form of protein G from which the albumin-binding region of the native molecule has been deleted genetically, thereby avoiding undesirable reactions with albumin. Recombinant protein G contains two Fc binding regions. Protein G Sepharose is a better choice for general purpose capture of antibodies since it binds a broader range of IgG from eukaryotic species and binds more classes of IgG. Usually protein G has a greater affinity than protein A for IgG and exhibits minimal binding to albumin, resulting in cleaner preparations and greater yields. The binding strength of protein G for IgG depends on the source species and subclass of the immunoglobulin. The dynamic binding capacity depends on the binding strength and also on several other factors, such as flow rate during sample application. Many antibodies also interact via the Fab region with a low affinity site on protein G. Protein G does not appear to bind human myeloma IgM, IgA or IgE, although some do bind weakly to protein A. Leakage of ligands from an affinity medium is always a possibility, especially if harsh elution conditions are used. The multi-point attachment of protein G to Sepharose results in very low leakage levels over a wide range of elution conditions. Purification options Binding capacity Maximum Comments operating flow HiTrap Human IgG, > 25 mg/column 4 ml/min (1 ml column) Purification of IgG, fragments and Protein G HP Human IgG, >125 mg/column 20 ml/min (5 ml column) subclasses, including human IgG3. Strong affinity for monoclonal mouse IgG1 and rat IgG. Prepacked columns. MAbTrap Kit Human IgG, > 25 mg/column 4 ml/min Purification of IgG, fragments and subclasses, including human IgG3. Strong affinity for monoclonal mouse IgG1 and rat IgG. Complete kit contains HiTrap Protein G HP (1 x 1 ml), accessories, pre-made buffers for 10 purifications and detailed experimental protocols. Protein G Human IgG, > 20 mg/ml medium 400 cm/h* Supplied as a suspension ready Sepharose 4 Cow IgG, 23 mg/ml medium for column packing. Fast Flow Goat IgG, 19 mg/ml medium Guinea pig IgG, 17 mg/ml medium Mouse IgG, 10 mg/ml medium Rat IgG, 7 mg/ml medium *See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.29 Purification examples Figure 11 shows the purification of mouse monoclonal IgG1 on HiTrap Protein G HP 1 ml. The monoclonal antibody was purified from a hybridoma cell culture supernatant. Sample: 12 ml mouse IgG1 hybridoma cell culture supernatant Column: HiTrap Protein G HP, 1 ml Flow: 1.0 ml/min Binding buffer: 20 mM sodium phosphate, pH 7.0 Elution buffer: 0.1 M glycine-HCI, pH 2.7 Electrophoresis: SDS-PAGE, PhastSystem™, PhastGel™ Gradient 10–15, 1 μl sample, silver stained Immunodiffusion: 1% Agarose A in 0.75 M Tris, 0.25 M 5,5-diethylbarbituric acid, 5 mM Ca-lactate, 0.02% sodium azide, pH 8.6 Lane 1. Low Molecular Weight Calibration Kit, reduced Lane 2. Mouse hybridoma cell culture fluid, non-reduced, diluted 1:10 Lane 3. Pool I, unbound material, non-reduced, diluted 1:10 Lane 4. Pool II, purified mouse IgG1, non-reduced, diluted 1:10 Lane 1 2 3 4 Mr SDS PAGE 14 000 20 100 30 000 45 000 66 000 97 000 Immunodiffusion A280 nm 5.0 2.5 0 5 10 15 20 25 30 ml pool I pool II Binding buffer Elution buffer Binding buffer Fig. 11. Purification of monoclonal mouse IgG1 on HiTrap Protein G HP, 1 ml. Figure 12 shows the purification of recombinant mouse Fab fragments, expressed in E. coli, using Protein G Sepharose 4 Fast Flow. Chimeric, non-immunogenic "humanized" mouse Fab, Fab' and F(ab')2 fragments are of great interest in tumour therapy since they penetrate tumours more rapidly and are also cleared from the circulation more rapidly than full size antibodies. Fig. 12. Purification of recombinant Fab fragments directed to the envelope protein gp120 of HIV-1 (anti-gp120 Fab), expressed in E. coli. Sample: Recombinant Fab fragment from E. coli. Medium: Protein G Sepharose 4 Fast Flow (1 ml) Flow: 0.2 ml/min (60 cm/h), or 0.3 ml/min (90 cm/h) Binding buffer: 0.15 M NaCl, 10 mM sodium phosphate, 10 mM EDTA, pH 7.0 Elution buffer: 0.5 M ammonium acetate, pH 3.0 Wash buffer: 1 M acetic acid, pH 2.5 0 0.5 1.5 2.5 3.5 0.0 Elution buffer UV 280 nm Conductivity pH 10.0 20.0 30.0 40.0 ml A280 nm30 Performing a separation Column: HiTrap Protein G HP, 1 ml or 5 ml Recommended flow rates: 1 ml/min (1 ml column) or 5 ml/min (5 ml column) Binding buffer: 0.02 M sodium phosphate, pH 7.0 Elution buffer: 0.1 M glycine-HCl, pH 2.7 Neutralization buffer: 1 M Tris-HCl, pH 9.0 Centrifuge samples (10 000 g for 10 minutes) to remove cells and debris. Filter through a 0.45 μm filter. If required, adjust sample conditions to the pH and ionic strength of the binding buffer either by buffer exchange on a desalting column or by dilution and pH adjustment (see page 134). 1. Equilibrate column with 5 column volumes of binding buffer. 2. Apply sample. 3. Wash with 5–10 column volumes of the binding buffer to remove impurities and unbound material. Continue until no protein is detected in the eluent (determined by UV absorbance at 280 nm). 4. Elute with 5 column volumes of elution buffer*. 5. Immediately re-equilibrate with 5–10 column volumes of binding buffer. *Since elution conditions are quite harsh, it is recommended to collect fractions into neutralization buffer (60 μl – 200 μl 1 M Tris-HCl, pH 9.0 per ml fraction), so that the final pH of the fractions will be approximately neutral. IgGs from most species and subclasses bind to protein G at near physiological pH and ionic strength. For the optimum binding conditions for IgG from a particular species, it is worth consulting the most recent literature. Avoid excessive washing if the interaction between the protein and the ligand is weak, since this may decrease the yield. Most immunoglobulin species do not elute from Protein G Sepharose until pH 2.7 or less. If biological activity of the antibody or antibody fragment is lost due to the low pH required for elution, try Protein A Sepharose: the elution pH may be less harsh. Desalt and/or transfer purified IgG fractions to a suitable buffer using a desalting column (see page 134). Reuse of Protein G Sepharose depends on the nature of the sample and should only be performed with identical samples to prevent cross-contamination. To increase capacity, connect several HiTrap Protein G HP columns (1 ml or 5 ml) in series. HiTrap columns can be used with a syringe, a peristaltic pump or connected to a liquid chromatography system, such as ÄKTAprime. For greater capacity pack a larger column with Protein G Sepharose 4 Fast Flow (see Appendix 3).31 MAbTrap Kit Fig. 13. MAbTrap Kit, ready for use. MAbTrap Kit contains a HiTrap Protein G HP 1 ml column, stock solutions of binding, elution and neutralization buffers, a syringe with fittings and an optimized purification protocol, as shown in Figure 13. The kit contains sufficient material for up to 20 purifications of monoclonal or polyclonal IgG from serum, cell culture supernatant or ascitic fluid, using a syringe. The column can also be connected to a peristaltic pump, if preferred. Figure 14 shows the purification of mouse monoclonal IgG1 from cell culture supernatant with syringe operation and a similar purification with pump operation. Eluted fractions were analysed by SDS-PAGE as shown in Figure 15. 3210A280 nm 1 4 7 10 13 16 19 22 25 28 31 ml A280 nm 3.0 2.0 1.0 0 5 10 15 20 25 30 ml Elution Column: HiTrap Protein G HP, 1 ml Sample: 10 ml mouse monoclonal cell supernatant, IgG1, anti-transferrin. Filtered through 0.45 μm filter Binding buffer: 20 mM sodium phosphate, pH 7.0 Elution buffer: 0.1 M glycine-HCl, pH 2.7 A) Syringe operation, approx. 60 drops/min B) Pump operation, flow 2 ml/min Fig. 14. Purification of mouse monoclonal IgG1 from cell culture supernatant. A. with syringe operation. B. with pump operation. Lanes 1 and 7. Low Molecular Weight Calibration Kit, Amersham Pharmacia Biotech Lane 2. Crude cell culture supernatant, mouse IgG1, diluted 1:11 Lane 3. Flow through, using a peristaltic pump, diluted 1:10 Lane 4. Eluted mouse IgG1, using a peristaltic pump Lane 5. Flow through, using a syringe, diluted 1:10 Lane 6. Eluted mouse IgG1, using a syringe 1 2 3 4 5 6 7 Mr 14 000 66 000 97 000 20 100 30 000 45 000 Fig. 15. SDS-PAGE on PhastSystem using PhastGel 10–15, non-reduced, and silver staining.32 Performing a separation Column: HiTrap Protein G HP, 1 ml Recommended flow rate: 1 ml/min Binding buffer: Dilute buffer concentrate 10-fold Elution buffer: Dilute buffer concentrate 10-fold Neutralization buffer: Add 60–200 μl of neutralization buffer per ml fraction to the test tubes in which IgG will be collected Centrifuge samples (10 000 g for 10 minutes) to remove cells and debris. Filter through a 0.45 μm filter. If required, adjust sample conditions to the pH and ionic strength of the binding buffer either by buffer exchange on a desalting column (see page 134) or by dilution and pH adjustment. Fig. 16. Using HiTrap Protein G HP with a syringe. A: Dilute buffers and prepare sample. Remove the column’s top cap and twist off the end. B: Equilibrate the column, load the sample and begin collecting fractions. C: Wash and elute, continuing to collect fractions. 1. Allow the column and buffers to warm to room temperature. 2. Dilute the binding and elution buffers. 3. Connect the syringe to the column using the luer adapter supplied. 4. Equilibrate the column with 5 ml distilled water, followed by 3 ml diluted binding buffer. 5. Apply the sample. 6. Wash with 5–10 ml diluted binding buffer until no material appears in the eluent. 7. Elute with 3–5 ml diluted elution buffer. Collect fractions into tubes containing neutralization buffer. 8. Immediately re-equilibrate the column with 5 ml diluted binding buffer. A B C33 Media characteristics Ligand Composition pH stability* Mean particle density size HiTrap Protein G HP 2 mg/ml Ligand coupled to Long term 3–9 34 μm (MAbTrap Kit) Sepharose HP by Short term 2–9 N-hydroxysuccinimide activation (gives stable attachment through alkylamine and ether links). Protein G Sepharose 4 2 mg/ml Ligand coupled to Long term 3–9 90 μm Fast Flow Sepharose 4 Fast Flow Short term 2–9 by cyanogen bromide activation. *Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. Chemical stability Stable in all common aqueous buffers. Storage Wash media and columns with 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C. HiTrap Protein A HP, Protein A Sepharose 4 Fast Flow, HiTrap rProtein A FF, rProtein A Sepharose 4 Fast Flow Protein A is derived from a strain of Staphylococcus aureus and contains five regions that bind to the Fc region of IgG. As an affinity ligand, protein A is coupled to Sepharose so that these regions are free to bind IgG. One molecule of protein A can bind at least two molecules of IgG. Both protein A and a recombinant protein A are available from Amersham Pharmacia Biotech. These molecules share similar specificities for the Fc region of IgG, but the recombinant protein A has been engineered to include a C-terminal cysteine that enables a single-point coupling to Sepharose. Single point coupling often results in an enhanced binding capacity. The binding strength of protein A for IgG depends on the source species of the immunoglobulin as well as the subclass of IgG (see Table 2). The dynamic binding capacity depends on the binding strength and also on several other factors, such as flow rate during sample application. Although IgG is the major reactive human immunoglobulin, some other types have also been demonstrated to bind to protein A. Interaction takes place with human colostral IgA as well as human myeloma IgA2 but not IgA1. Some human monoclonal IgMs and some IgMs from normal and macroglobulinaemic sera can bind to protein A. Leakage of ligands from an affinity medium is always a pos