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Sample clarification Centrifugation and filtration are standard laboratory techniques for sample clarification and are used routinely when handling small samples. It is highly recommended to centrifuge and filter any sample immediately before chromatographic purification. Centrifugation Centrifugation removes lipids and particulate matter, such as cell debris. If the sample is still not clear after centrifugation, use filter paper or a 5 µm filter as a first step and one of the filters below as a second step filter. • For small sample volumes or proteins that adsorb to filters, centrifuge at 10 000 g for 15 minutes. • For cell lysates, centrifuge at 40 000–50 000 g for 30 minutes. • Serum samples can be filtered through glass wool after centrifugation to remove any remaining lipids. Filtration Filtration removes particulate matter. Membrane filters that give the least amount of nonspecific binding of proteins are composed of cellulose acetate or PVDF. For sample preparation before chromatography, select a filter pore size in relation to the bead size of the chromatographic medium. Nominal pore size of filter Particle size of chromatographic medium 1 µm 90 µm and upwards 0.45 µm 34 µm 0.22 µm 3, 10, 15 µm or when extra clean samples or sterile filtration is required Check the recovery of the target protein in a test run. Some proteins may adsorb nonspecifically to filter surfaces. Desalting Desalting columns are suitable for any sample volume and will rapidly remove low molecular weight contaminants in a single step at the same time as transferring the sample into the correct buffer conditions. Centrifugation and/or filtration of the sample before desalting is still recommended. Detailed procedures for buffer exchange and desalting are given on page 134. At laboratory scale, when samples are reasonably clean after filtration or centrifugation, the buffer exchange and desalting step can be avoided. For affinity chromatography or hydrophobic interaction chromatography, it may be sufficient to adjust the pH of the sample and, if necessary, dilute to reduce the ionic strength of the solution. Rapidly process small or large sample volumes. Use before and/or between purification steps, if needed (remember that each extra step can reduce yield and desalting also dilutes the sample). 130
Remove salts from proteins with molecular weight M r > 5 000. Use 100 mM ammonium acetate or 100 mM ammonium hydrogen carbonate if volatile buffers are required. Specific sample preparation steps Specific sample preparation steps may be required if the crude sample is known to contain contamininants such as lipids, lipoproteins or phenol red that may build up on a column or if certain gross impurities, such as bulk protein, should be removed before any chromatographic step. Fractional precipitation Fractional precipitation is frequently used at laboratory scale to remove gross impurities from small sample volumes, and occasionally used in small-scale commercial production. Precipitation techniques separate fractions by the principle of differential solubility. Because protein species differ in their degree of hydrophobicity, increased salt concentrations can enhance hydrophobic interactions between the proteins and cause precipitation. Fractional precipitation can be applied to remove gross impurities in three different ways, as shown in Figure 74. Clarification Bulk proteins and particulate matter precipitated Supernatant Extraction Clarification Concentration Target protein precipitated with proteins of similar solubility Extraction Clarification Bulk proteins and particulate matter precipitated Concentration Target protein precipitated with proteins of similar solubility Redissolve pellet* Chromatography Remember: if precipitating agent is incompatible with next purification step, TM use Sephadex G-25 for desalting and buffer exchange e.g. HiTrap Desalting or PD-10 columns Redissolve pellet* *Remember: not all proteins are easy to redissolve, yield may be reduced Fig. 74. Three ways to use precipitation. 131
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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
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pH elution A change in pH alters th
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0.6 0.4 0.2 0 A280 pH selected for
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Situation Cause Remedy Protein does
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Chapter 3 Purification of specific
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Table 2. Relative binding strengths
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Purification examples Figure 11 sho
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MAbTrap Kit Fig. 13. MAbTrap Kit, r
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Media characteristics Ligand Compos
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A 280 nm 2.0 1.5 1.0 0.5 Column: rP
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Desalt and/or transfer purified IgG
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Performing a separation Column: Rec
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M r 97 000 66 000 45 000 30 000 20
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in The Recombinant Protein Handbook
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Cleaning These procedures are appli
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Purification examples Figures 24 an
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Nickel solution: 0.1 M NiSO 4 Bindi
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Protein A fusion proteins IgG Sepha
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Purification or removal of serine p
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Sample: 2 ml clarified E. coli homo
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Serine proteases and zymogens with
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DNA binding proteins HiTrap Heparin
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Sample: Column: Binding buffer: Elu
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Media characteristics Ligand densit
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Sample: Pooled and frozen human pla
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The harsh conditions required to br
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Purification or removal of albumin
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IFN-act. A units 280 nm 250 0.12 20
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Purification options Binding capaci
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Sepharose NH (CH 2) n NH N N O N N
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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
Page 123 and 124: Ligands containing amino groups can
Page 125 and 126: Chapter 6 Affinity chromatography a
Page 127 and 128: Resolution is achieved by the selec
Page 129 and 130: For any capture step, select the te
Page 131: Appendix 1 Sample preparation Sampl
Page 135 and 136: 1. Filter (0.45 µm) or centrifuge
Page 137 and 138: For laboratory scale operations, Ta
Page 139 and 140: Removal of lipoproteins Lipoprotein
Page 141 and 142: Appendix 2 Selection of purificatio
Page 143 and 144: 4. Estimate the amount of slurry (r
Page 145 and 146: Appendix 5 Conversion data: protein
Page 147 and 148: Middle unit residue (-H 2 0) Charge
Page 149 and 150: Expected results when changing cond
Page 151 and 152: Expected results with competitive e
Page 153 and 154: Appendix 8 Analytical assays during
Page 155 and 156: Appendix 9 Storage of biological sa
Page 157 and 158: Ordering information Product Quanti
Page 159 and 160: STREAMLINE, Sepharose, HiTrap, ÄKT
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