Affinity Chromatography - Department of Molecular and Cellular ...

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Purification options Binding capacity Maximum Comments operating flow HiTrap Human IgG, > 20 mg/column 4 ml/min (1 ml column) Purification of IgG, fragments Protein A HP Human IgG, > 100 mg/column 20 ml/min (5 ml column) and sub-classes. Prepacked columns. Protein A Human IgG, > 35 mg/ml medium 400 cm/h** Supplied as a suspension Sepharose 4 Mouse IgG, 3–10 mg/ml medium ready for column packing. Fast Flow* HiTrap Human IgG, > 50 mg/column 4 ml/min (1 ml column) Purification of IgG, fragments rProtein A FF Human IgG, > 250 mg/column 20 ml/min (5 ml column) and sub-classes. Enhanced binding capacity. Prepacked columns. rProtein A Human IgG, > 50 mg/ml medium 300 cm/h** Enhanced binding capacity. Sepharose 4 Mouse IgG, 8–20 mg/ml medium Supplied as a suspension Fast Flow* ready for column packing. *Protein A Sepharose 4 Fast Flow and rProtein A Sepharose Fast Flow have a higher binding capacity, a more rigid matrix and provide more convenient alternatives to Protein A Sepharose CL-4B, which must be rehydrated before column packing. **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. Purification examples Figure 17 shows the purification of mouse IgG 2b from ascites fluid on HiTrap rProtein A FF 1 ml column using a syringe. The eluted pool contained 1 mg IgG 2b and the silver stained SDS-PAGE gel confirmed a purity level of over 95%. Sample: 1 ml of mouse ascites containing IgG 2b , filtered through a 0.45 µm filter. The sample was a kind gift from Dr. N. Linde, EC Diagnostics, Sweden Column: HiTrap rProtein A FF 1 ml Binding buffer: 0.02 M sodium phosphate, pH 7.0 Elution buffer: 0.1 M sodium citrate, pH 3.0 Flow: ~ 1 ml/min A 280 nm 2.5 2.0 1.5 1.0 0.5 Elution buffer 0.0 0 2 4 6 8 10 12 14 16Volume (ml) Time (min) Flow-through Eluted IgG 2b pool pool M r 97 000 66 000 45 000 30 000 20 100 14 000 1 2 3 4 IgG 2b SDS-PAGE on PhastSystem using PhastGel Gradient 10–15, silver staining Lane 1. Low Molecular Weight Calibration Kit, Amersham Pharmacia Biotech Lane 2. Starting material, diluted 10-fold Lane 3. Flow-through pool Lane 4. Eluted IgG 2b pool Fig. 17. Purification of mouse IgG 2b from ascites on HiTrap rProtein A FF 1 ml column using a syringe. Figure 18 shows a larger scale purification of monoclonal mouse IgG 2a from a clarified hybridoma cell culture on rProtein A Sepharose Fast Flow. Sample loading was over 9 mg IgG/ml of medium, with a 95% recovery of highly purified antibody. 34
A 280 nm 2.0 1.5 1.0 0.5 Column: rProtein A Sepharose Fast Flow, XK 16/20, bed height 4.8 cm (9.6 ml) Sample: 600 ml clarified cell culture containing 87.6 mg IgG 2a Binding buffer: 20 mM sodium phosphate, pH 7.0 Elution buffer: 20 mM sodium citrate, pH 4.0 Flow: 5 ml/min (150 cm/h) M r 97 000 66 000 45 000 30 000 20 100 14 400 0.0 0 200 400 600 Volume (ml) 1 2 3 4 Fig. 18a. Purification of a monoclonal IgG 2a from clarified cell culture on rProtein A Sepharose 4 Fast Flow. Fig. 18b. SDS-PAGE of starting material (lane 2) and eluate (lane 3). The samples were concentrated 10 times and reduced. Lane 1 and 4 are LMW markers. PhastSystem, PhastGel Gradient 10–15. Performing a separation Column: HiTrap Protein A HP, 1 ml or 5 ml, or HiTrap rProtein A FF, 1 ml or 5 ml Recommended flow rates: 1 ml/min (1 ml columns) or 5 ml/min (5 ml columns) Binding buffer: 0.02 M sodium phosphate, pH 7.0 Elution buffer: 0.1 M citric acid, pH 3–6 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 needed, 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. A HiTrap column can be used with a syringe, a peristaltic pump or connected to a liquid chromatography system, such as ÄKTAprime. 1. Equilibrate the 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, 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. 35
Page 1 and 2: Affinity Chromatography Principles
Page 3 and 4: Affinity Chromatography Principles
Page 5 and 6: Serine proteases and zymogens with
Page 7: Appendix 4 ........................
Page 10 and 11: This handbook describes the role of
Page 12 and 13: Affinity chromatography is also use
Page 14 and 15: BioProcess Media for large-scale pr
Page 17 and 18: Chapter 2 Affinity chromatography i
Page 19 and 20: Avoid using magnetic stirrers as th
Page 21 and 22: pH elution A change in pH alters th
Page 23 and 24: 0.6 0.4 0.2 0 A280 pH selected for
Page 25 and 26: Situation Cause Remedy Protein does
Page 27 and 28: Chapter 3 Purification of specific
Page 29 and 30: Table 2. Relative binding strengths
Page 31 and 32: Purification examples Figure 11 sho
Page 33 and 34: MAbTrap Kit Fig. 13. MAbTrap Kit, r
Page 35: Media characteristics Ligand Compos
Page 39 and 40: Desalt and/or transfer purified IgG
Page 41 and 42: Performing a separation Column: Rec
Page 43 and 44: M r 97 000 66 000 45 000 30 000 20
Page 45 and 46: in The Recombinant Protein Handbook
Page 47 and 48: Cleaning These procedures are appli
Page 49 and 50: Purification examples Figures 24 an
Page 51 and 52: Nickel solution: 0.1 M NiSO 4 Bindi
Page 53 and 54: Protein A fusion proteins IgG Sepha
Page 55 and 56: Purification or removal of serine p
Page 57 and 58: Sample: 2 ml clarified E. coli homo
Page 59 and 60: Serine proteases and zymogens with
Page 61 and 62: DNA binding proteins HiTrap Heparin
Page 63 and 64: Sample: Column: Binding buffer: Elu
Page 65 and 66: Media characteristics Ligand densit
Page 67 and 68: Sample: Pooled and frozen human pla
Page 69 and 70: The harsh conditions required to br
Page 71 and 72: Purification or removal of albumin
Page 73 and 74: IFN-act. A units 280 nm 250 0.12 20
Page 75 and 76: Purification options Binding capaci
Page 77 and 78: Sepharose NH (CH 2) n NH N N O N N
Page 79 and 80: If detergent or denaturing agents h
Page 81 and 82: Glycoproteins or polysaccharides Co
Page 83 and 84: For complex samples containing glyc
Page 85 and 86: For complex samples containing glyc
Page 87 and 88:
Chemical stability Avoid exposure t
Page 89 and 90:
Proteins and peptides with exposed
Page 91 and 92:
Several methods can be used to dete
Page 93 and 94:
Media characteristics Composition M
Page 95 and 96:
Performing a separation Binding buf
Page 97 and 98:
Do not store the suspension for lon
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Sepharose has been modified and dev
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Ligand coupling Several methods are
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Couple a ligand through the least c
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Table 9 summarizes recommended liga
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Coupling through the primary amine
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Ligand and column preparation 1. Di
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Options Product Spacer Coupling con
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100 80 Coupled substance, % 60 40 2
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Coupling small ligands through amin
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Ligand coupling 1. Add the ligand s
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Alternative coupling solutions: Dis
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Media characteristics Product Compo
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Ligands containing amino groups can
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Chapter 6 Affinity chromatography a
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Resolution is achieved by the selec
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For any capture step, select the te
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Appendix 1 Sample preparation Sampl
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Remove salts from proteins with mol
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1. Filter (0.45 µm) or centrifuge
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For laboratory scale operations, Ta
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Removal of lipoproteins Lipoprotein
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Appendix 2 Selection of purificatio
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4. Estimate the amount of slurry (r
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Appendix 5 Conversion data: protein
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Middle unit residue (-H 2 0) Charge
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Expected results when changing cond
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Expected results with competitive e
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Appendix 8 Analytical assays during
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Appendix 9 Storage of biological sa
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Ordering information Product Quanti
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STREAMLINE, Sepharose, HiTrap, ÄKT
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