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Purification examples Figure 41 shows the use of HiTrap Blue HP for purification of increasing amounts of human serum albumin. The process is easily scaled up by connecting several 1 ml or 5 ml HiTrap columns is series. In this example elution is achieved by increasing the ionic strength of the buffer. Changing the pH or the polarity of the buffer can also work. Figure 42 shows the use of Blue Sepharose 6 Fast Flow for the separation of human serum albumin from interferon b. Sample: Human serum buffer exchanged on a PD-10 Desalting column to binding buffer. Filtered on a 0.45 µm filter Column: HiTrap Blue HP, 1 ml or 5 ml Flow: 2 ml/min (1 ml column), 4 ml/min (5 ml column) Binding buffer: 50 mM KH 2 PO 4 , pH 7.0 Elution buffer: 50 mM KH 2 PO 4 , 1.5 M KCl, pH 7.0 Configuration: 1×1 ml column Sample vol.: 0.7 ml human serum Yield: 16.7 mg HSA Configuration: 2×1 ml column Sample vol.: 1.4 ml human serum Yield: 33.2 mg HSA Configuration: 3×1 ml column Sample vol.: 2.1 ml human serum Yield: 52.0 mg HSA A 280 nm 1.0 Binding buffer Elution buffer A 280 nm 1.0 Binding buffer Fractions 2–10 Elution buffer Fractions 16–21 A 280 nm 1.0 Binding buffer Fractions 2–10 Elution buffer Fractions 17–22 Fractions 2–10 Fractions 15–20 10 20 ml 10 20 ml 10 20 ml Configuration: 1×5 ml column Sample vol.: 3.5 ml human serum Yield: 98.5 mg HSA Configuration: 3×5 ml columns connected in series Sample vol.: 10.5 ml human serum Yield: 286.9 mg HSA A 280 nm A 280 nm Binding buffer Elution buffer Binding buffer Elution buffer Fractions 22–30 Fractions 67–83 1.0 Fractions 3–14 1.0 Fractions 11–46 10 20 30 ml 10 20 30 40 50 60 70 80 90 100 110 ml Fig. 41. Scaling up on HiTrap Blue HP gives predictable separations and quantitatively reproducible yields. 70
IFN-act. A units 280 nm 250 0.12 200 150 100 IFNactivity A 280nm 0.10 0.08 0.06 0.04 Sample: 0.5 ml interferon b (1 000 000 U/ml) in 0.1 M phosphate, pH 7.4, with 1 mg/ml of human serum albumin Column: Blue Sepharose 6 Fast Flow (0.5 ml) Flow: Gravity feed Binding buffer: 0.02 M phosphate, 0.15 M NaCl, pH 7.2 Elution buffer 1: 0.02 M phosphate, 2 M NaCl, pH 7.2 Elution buffer 2: 0.02 M phosphate, 2 M NaCl, 50% ethylene glycol, pH 7.2 50 0.02 0 0 5 10 Volume (ml) 0 Elution buffer 1 Elution buffer 2 Fig. 42. Purification of human serum albumin and interferon b on Blue Sepharose 6 Fast Flow. In these examples elution is achieved by increasing the ionic strength of the buffer or changing the polarity of the buffer. Changing the pH of the buffer can also work, but the correct co-factor is preferable for the elution of specifically bound proteins. Performing a separation Binding buffer: 50 mM KH 2 PO 4 , pH 7.0 or 20 mM sodium phosphate, pH 7.0 Elution buffer: 50 mM KH 2 PO 4 , 1.5 M KCl, pH 7.0 or 20 mM sodium phosphate, 2 M NaCl, pH 7.0 1. Equilibrate the column with 5 column volumes of binding buffer. 2. Apply the sample, using a syringe or a pump. 3. Wash with 10 column volumes of binding buffer or until no material appears in the eluent (monitored by absorption at A 280 nm ). 4. Elute with 5 column volumes of elution buffer. More may be required if the interaction is difficult to reverse. Cleaning Wash with 5 column volumes of high pH (0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5) followed by low pH (0.1 M sodium acetate, 0.5 M NaCl, pH 4.5). Repeat 4–5 times. Re-equilibrate immediately with binding buffer. Remove precipitated proteins with 4 column volumes of 0.1 M NaOH at a low flow rate, followed by washing with 3–4 column volumes of 70% ethanol or 2 M potassium thiocyanate. Alternatively, wash with 2 column volumes of 6 M guanidine hydrochloride. Re-equilibrate immediately with binding buffer. Remove strongly hydrophobic proteins, lipoproteins and lipids by washing with 3–4 column volumes of up to 70% ethanol or 30% isopropanol. Alternatively, wash with 2 column volumes of detergent in a basic or acidic solution, e.g. 0.1% non-ionic detergent in 1 M acetic acid at a low flow rate, followed by 5 column volumes of 70% ethanol to remove residual detergent. Re-equilibrate immediately in binding buffer. 71
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Affinity Chromatography Principles
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Affinity Chromatography Principles
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Serine proteases and zymogens with
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Appendix 4 ........................
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This handbook describes the role of
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Affinity chromatography is also use
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BioProcess Media for large-scale pr
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Chapter 2 Affinity chromatography i
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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 and 36: Media characteristics Ligand Compos
Page 37 and 38: A 280 nm 2.0 1.5 1.0 0.5 Column: rP
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: Purification or removal of albumin
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
Page 99 and 100: Sepharose has been modified and dev
Page 101 and 102: Ligand coupling Several methods are
Page 103 and 104: Couple a ligand through the least c
Page 105 and 106: Table 9 summarizes recommended liga
Page 107 and 108: Coupling through the primary amine
Page 109 and 110: Ligand and column preparation 1. Di
Page 111 and 112: Options Product Spacer Coupling con
Page 113 and 114: 100 80 Coupled substance, % 60 40 2
Page 115 and 116: Coupling small ligands through amin
Page 117 and 118: Ligand coupling 1. Add the ligand s
Page 119 and 120: Alternative coupling solutions: Dis
Page 121 and 122: 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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