Affinity chromatography is also used to remove specific contaminants,for example Benzamidine SepharoseTM 6 Fast Flow can remove serine proteases,such as thrombin and Factor Xa.Figure 2 shows the key stages in an affinity purification. 1.Affinity medium is equilibrated in binding buffer. ng ot the sto a comp through the column. 4.Affinity medium isrequilrated with binding buffer. bration emen 12c 12 Fig.2.Typical affinity purification. 10
10 Column Volumes (cv) begin sample application change to elution buffer x cv 1-2 cv >1 1-2 cv 1-2 cv 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 favor 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 favor 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
one simple step,inlud ding,for example,commc on 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 HiTrapTM 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 AKTATMdesign chromatography system.Several HiTrap columns can be connected in series to increase purification capacity and all columns are supplied with detailed protocols for use. 包o Table 1.HiTrap and HiPrep affinity columns for laboratory al purification. Application HiTrap and HiPrep columns Isolation of human immunoglobulins IgG.fragments and subclasses HiTran rProteio A FF 1 ml and 5ml leG fraements and subclasse HiTrap Protein A HP.1 ml and 5 m includi wan lgY from egg yol HiTrap gY Purification HP.5m Mouse and human lgM HiTrap IgM Purification HP.1 ml (His)s fusion proteins nHP1mland5 GST tusion roteins ns and peptides with exposed His.Cys or Trp Trap Chelaing HP.1mland 5ml Biotinylated substances HiTrap Streptavidin HP.1m ONA binding proteins and coagulation factors Trypsin-like serine proteases including Factor Xa,thrombin and trypsin FF(hgh u) Matrix for affinity media.Coupling via primary amine HiTrap NHS-activated HP
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 GSTPrep™ FF 16/10, 20 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 ml
BioProcess Media for large-scale production BioProc Specific BioProce Media have been designed for each chom &raphi stage in a process m Capture to P with clear place,at the right time.Amersham Biosciences 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 process validation,as well as providing support for submissions to regulatory uthorities.Using BioPro ess media fo High flow rate,high capa city and high covery co bute to the omy industrial process All BioProcess Media have chemical stability to allow efficient cleaning and sanitizatio s are established fo r a wide range of scales and compatibl Custom Designed Media and Columns Prepacked columns,made according to the client's choice from the Amersham Biosciences range of columns and media,can be supplied by the Custom Products Group. Custom Designed Media(CDM)can be produced for specific industrial process separation when suitable edia the stande ge.The CDM Am test and deliv ration with the to des gn,n fac separation requirements.Whe ograph step is developed to be an integral part of a mar ring process,the che ice o column i important to nsure consistent performance and reliable operation.Amersham Biosciences 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
12 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 Biosciences 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 Catalog from Amersham Biosciences 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 Biosciences 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 Biosciences 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 Biosciences 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
Common terms in affinity chromatography 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 toa specific target molecule or group of target molecules. Binding:buffer conditions are optimized to ensure that the target molecules interact 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. 13
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 chromatography
Chapter 2 Affinity chromatography in practice purificati 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 Biosciences 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 0 the gand and unbound a through the column. n a purified,concentra 4.Affinity medium is re-quilbrated with binding buffer. re-eguilibratioe 126 Fig.3.Typical affinity purification
15 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 Biosciences 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 favor 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 favor 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 1-2 cv 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