Chapter 25 Chromatographic Techniques in the Downstream Processing of Proteins in Biotechnology Ruth Freitag Abstract The purification of the product, the so-called downstream process (DSP), tends to be one of the most costly aspects of modern bioprocessing, especially in the case of proteins. In such cases, chromatography is still the major tool on all levels of the DSP from the first capture to the final polishing step. In this chapter, we will first outline the commonly used methods and their setup, in particular ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), affinity chromatography (AC), and gel filtration (GPC, SEC), but also some less-known alternatives such as hydroxyapatite chromatography (HAC). Then the rational design of a downstream process, which usually comprises three orthogonal chromatographic steps, is discussed. Finally, process variants deviating from the usual batch-column/gradient elution approach will be presented, including expanded bed, displacement, and continuous chromatography, but also affinity precipitation. A most recent trend observable in the biotechnical DSP is the drive towards disposable elements (single-use technologies). Some options for this will be discussed as well. Key words Affinity, Capture, Chromatography, DSP, GPC, HIC, IEX, Isolation, Protein, Purification, SEC
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Introduction Biotechnical products today find many uses, from biopharmaceuticals in medicine to technical biocatalysts in the chemical industry or as tools in sustainable energy provision and bioremediation. Many of these products are proteins; often they are produced recombinantly, i.e., by genetically modified organisms. Prior to use, they need to be recovered and purified. The required final degree of purity may vary from >99 % in a biopharmaceutical intended for injection to pH 9). In addition, for many of these columns, a HPLC apparatus may be required for operation due to the high backpressures of the columns. 13. The TFA (trifluoroacetic acid) acts as an ion-pairing agent (increasing the hydrophobicity/retention of the protein) and also shields residual charges on the stationary phase surface that may otherwise give rise to secondary electrostatic interactions. 1 % phosphoric acid or triethylammonium phosphate may be used alternatively. 14. Acetonitrile is a very common modifier in RPC. Methanol and isopropanol can be used instead and with some columns/ applications are to be preferred. See the manufacturer’s instructions (application notes) for hints in this regard. 15. Hydroxyapatite becomes unstable below a pH of 6 but can be treated (cleaned/sanitized) with 1 M NaOH. Recently a fluoroapatite material has become available for protein chromatography from BioRad that can be used down to a pH of 5. 16. Hydroxyapatite (HA) shows a mixed-mode interaction with proteins including both electrostatic interaction with positively charged substances and C-site interaction, e.g., with proteins bearing carboxylic acid (chelating interaction) or phosphate groups on the surface [9]. The buffer’s pH and composition will influence the relative importance of these two types of interaction for a given protein. Buffers in HA chromatography usually have near neutral pH, but values as low as 6 and as high as 9 (Tris–HCl buffer) have been reported. Elution in HA chromatography is usually done in a gradient of increasing phosphate concentration, which diminishes both types of interaction, P-site interaction due to charge screening and C-site interaction due to competitive interaction of the buffer’s phosphate ions with the C-sites. However, especially for proteins interacting predominately by electrostatic interaction (P-sites), elution in a NaCl gradient (up to 1 M) is also possible, especially when prepared in a low concentration phosphate background buffer (this will suppress any residual C-site interaction). Antibodies have, e.g., been known to elute readily in a NaCl gradient prepared in a 20 mM phosphate buffer, while no elution by NaCl was possible in the absence of phosphate [10, 24].
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17. The displacer concentration influences the concentration in the substance zones (see also Note 54) and must be adjusted accordingly. 18. In general, this means that the displacer elutes after the target proteins in an analytical chromatographic separation, i.e., shows higher retention under gradient conditions. However, this may not be the case for substances with crossing isotherms. Recording the respective single substance isotherms, e.g., according to [16], is strongly suggested for a full characterization of the system. 19. In addition, small molecules have been developed as displacer for a variety of chromatographic stationary phases and modes, see, e.g., [25, 26]. In the case of hydroxyapatite displacement chromatography EGTA (ethylene-glycol-bis-(β-aminoethyl ether)-N,N,N′,N′ acetic acid) has been suggested as displacer of C-site interacting substances [27], while P-site interacting substances may be displaced by most standard cation exchange displacers. 20. Affinity precipitation depends on the reversible precipitation of the AML/target protein-affinity complex once a certain critical temperature has been surpassed. The critical temperature depends on the solution and is, e.g., lowered by most salts [28]. For any given buffer to be used in affinity precipitation, the critical solution temperature should therefore be determined (see also Note 66). 21. 280 nm detects the aromatic amino acids. Proteins that are poor in these amino acids give a comparatively weak signal at this wavelength. A detection wavelength of 214 nm (peptide bond) is more generic and often more—sometimes too—sensitive. 22. Column and system flushing is especially important in biochromatography, where typically aqueous, salt-containing mobile phases are used. Salt crystals forming after water evaporation can quickly damage pumps and columns. 23. Instead of binding the target molecule to the ion exchanger, it is also possible to selectively bind contaminants and impurities and let the product run through the column (negative chromatography). An anion exchanger may, e.g., be used to selectively remove anionic contaminants (DNA, endotoxins, anionic proteins) from a cationic product, e.g., an antibody. 24. If the isoelectric point of the target molecule is not known, choose either a strong anion exchanger at pH 8 (Tris–HCl buffer) for binding IEX or a strong cation exchanger at pH 8 for nonbinding conditions to start the method development (see also Note 23). In case of cation exchange chromatography, a 20 mM phosphate buffer (pH 6) is a good starting point for binding conditions. The stationary phase material is also of importance. Even if the same type of interactive group (i.e., a
Protein Chromatography
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series of Q-materials) is used, differences in protein adsorption will be observed, mostly due to secondary interactions with the stationary phase matrix. Making the best choice can therefore be difficult. However, often a standard column or column selection is already in use in the research group. This is a good starting point. If this is not the case, many established providers of chromatographic materials also sell sets of small, prepacked columns of their standard ion exchanger materials, which can also provide a good basis for column scouting. If at all possible, prepacked columns should be used for scouting to avoid the challenge/bias of column packing. If a stationary phase is only available in loose form, packing instructions can usually be obtained from the supplier. Empty columns for packing can be obtained from the supplier of the chromatographic system. 25. Keep in mind that the net charge of a protein represents the sum over all charges. Even a nominally negatively charged protein will still contain a number of positively charged amino acid residues. Moreover, most proteins dissolve least well at their isoelectric point. 26. During column loading, conditions are usually chosen in such a way that the target molecules are strongly bound. In terms of column loading, the sample volume is therefore less important than the total amount of protein in the feed. The dynamic protein binding capacity is provided by the supplier of the column or can be determined by measuring the breakthrough curve. For method scouting, column loading should not be too heavy. For an optimized separation up to 80 % of the dynamic protein capacity can sometimes be used in gradient elution approaches. 27. By dividing the volumetric flow rate (mL/min) by the column’s cross-sectional area (in cm2), the linear flow rate (cm/min) is obtained. The linear flow rate is kept constant during column scale-up. 28. Any real-life protein feed even after clarification (filtration, 0.45 μm) may still contain components that can harm the column. A guard column is a small column packed from the same stationary phase as the separation column and inserted into the flow line just ahead of the separation column. Most of the damage is then done in the guard column, which is regularly replaced after a few cycles. 29. Instead of regeneration with pure Buffer B, it is possible to regenerate with a higher salt buffer (e.g., 2 M NaCl in Buffer A). 30. A conductivity detector is a good means to see when the high salt buffer has been completely displaced from the column during reequilibration.
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31. When increasing the flow rate, keep the pressure limit of the column and other system components (detector cell!) in mind. 32. The ability of a salt to enforce hydrophobic interaction is linked to its position in the Hofmeister series [29]. Sodium sulfate is a good alternative to ammonium sulfate. NaCl is a comparatively weak enforcer of hydrophobic interactions. 33. Column selection is not always straightforward in HIC as the hydrophobicity of a protein is difficult to predict. Very hydrophobic proteins need mildly hydrophobic stationary phases else the interaction becomes too strong (difficult elution) and vice versa. Binding strength will increase with ligands in the following order: ether
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Introduction Biotechnical products today find many uses, from biopharmaceuticals in medicine to technical biocatalysts in the chemical industry or as tools in sustainable energy provision and bioremediation. Many of these products are proteins; often they are produced recombinantly, i.e., by genetically modified organisms. Prior to use, they need to be recovered and purified. The required final degree of purity may vary from >99 % in a biopharmaceutical intended for injection to pH 9). In addition, for many of these columns, a HPLC apparatus may be required for operation due to the high backpressures of the columns. 13. The TFA (trifluoroacetic acid) acts as an ion-pairing agent (increasing the hydrophobicity/retention of the protein) and also shields residual charges on the stationary phase surface that may otherwise give rise to secondary electrostatic interactions. 1 % phosphoric acid or triethylammonium phosphate may be used alternatively. 14. Acetonitrile is a very common modifier in RPC. Methanol and isopropanol can be used instead and with some columns/ applications are to be preferred. See the manufacturer’s instructions (application notes) for hints in this regard. 15. Hydroxyapatite becomes unstable below a pH of 6 but can be treated (cleaned/sanitized) with 1 M NaOH. Recently a fluoroapatite material has become available for protein chromatography from BioRad that can be used down to a pH of 5. 16. Hydroxyapatite (HA) shows a mixed-mode interaction with proteins including both electrostatic interaction with positively charged substances and C-site interaction, e.g., with proteins bearing carboxylic acid (chelating interaction) or phosphate groups on the surface [9]. The buffer’s pH and composition will influence the relative importance of these two types of interaction for a given protein. Buffers in HA chromatography usually have near neutral pH, but values as low as 6 and as high as 9 (Tris–HCl buffer) have been reported. Elution in HA chromatography is usually done in a gradient of increasing phosphate concentration, which diminishes both types of interaction, P-site interaction due to charge screening and C-site interaction due to competitive interaction of the buffer’s phosphate ions with the C-sites. However, especially for proteins interacting predominately by electrostatic interaction (P-sites), elution in a NaCl gradient (up to 1 M) is also possible, especially when prepared in a low concentration phosphate background buffer (this will suppress any residual C-site interaction). Antibodies have, e.g., been known to elute readily in a NaCl gradient prepared in a 20 mM phosphate buffer, while no elution by NaCl was possible in the absence of phosphate [10, 24].
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Ruth Freitag
17. The displacer concentration influences the concentration in the substance zones (see also Note 54) and must be adjusted accordingly. 18. In general, this means that the displacer elutes after the target proteins in an analytical chromatographic separation, i.e., shows higher retention under gradient conditions. However, this may not be the case for substances with crossing isotherms. Recording the respective single substance isotherms, e.g., according to [16], is strongly suggested for a full characterization of the system. 19. In addition, small molecules have been developed as displacer for a variety of chromatographic stationary phases and modes, see, e.g., [25, 26]. In the case of hydroxyapatite displacement chromatography EGTA (ethylene-glycol-bis-(β-aminoethyl ether)-N,N,N′,N′ acetic acid) has been suggested as displacer of C-site interacting substances [27], while P-site interacting substances may be displaced by most standard cation exchange displacers. 20. Affinity precipitation depends on the reversible precipitation of the AML/target protein-affinity complex once a certain critical temperature has been surpassed. The critical temperature depends on the solution and is, e.g., lowered by most salts [28]. For any given buffer to be used in affinity precipitation, the critical solution temperature should therefore be determined (see also Note 66). 21. 280 nm detects the aromatic amino acids. Proteins that are poor in these amino acids give a comparatively weak signal at this wavelength. A detection wavelength of 214 nm (peptide bond) is more generic and often more—sometimes too—sensitive. 22. Column and system flushing is especially important in biochromatography, where typically aqueous, salt-containing mobile phases are used. Salt crystals forming after water evaporation can quickly damage pumps and columns. 23. Instead of binding the target molecule to the ion exchanger, it is also possible to selectively bind contaminants and impurities and let the product run through the column (negative chromatography). An anion exchanger may, e.g., be used to selectively remove anionic contaminants (DNA, endotoxins, anionic proteins) from a cationic product, e.g., an antibody. 24. If the isoelectric point of the target molecule is not known, choose either a strong anion exchanger at pH 8 (Tris–HCl buffer) for binding IEX or a strong cation exchanger at pH 8 for nonbinding conditions to start the method development (see also Note 23). In case of cation exchange chromatography, a 20 mM phosphate buffer (pH 6) is a good starting point for binding conditions. The stationary phase material is also of importance. Even if the same type of interactive group (i.e., a
Protein Chromatography
449
series of Q-materials) is used, differences in protein adsorption will be observed, mostly due to secondary interactions with the stationary phase matrix. Making the best choice can therefore be difficult. However, often a standard column or column selection is already in use in the research group. This is a good starting point. If this is not the case, many established providers of chromatographic materials also sell sets of small, prepacked columns of their standard ion exchanger materials, which can also provide a good basis for column scouting. If at all possible, prepacked columns should be used for scouting to avoid the challenge/bias of column packing. If a stationary phase is only available in loose form, packing instructions can usually be obtained from the supplier. Empty columns for packing can be obtained from the supplier of the chromatographic system. 25. Keep in mind that the net charge of a protein represents the sum over all charges. Even a nominally negatively charged protein will still contain a number of positively charged amino acid residues. Moreover, most proteins dissolve least well at their isoelectric point. 26. During column loading, conditions are usually chosen in such a way that the target molecules are strongly bound. In terms of column loading, the sample volume is therefore less important than the total amount of protein in the feed. The dynamic protein binding capacity is provided by the supplier of the column or can be determined by measuring the breakthrough curve. For method scouting, column loading should not be too heavy. For an optimized separation up to 80 % of the dynamic protein capacity can sometimes be used in gradient elution approaches. 27. By dividing the volumetric flow rate (mL/min) by the column’s cross-sectional area (in cm2), the linear flow rate (cm/min) is obtained. The linear flow rate is kept constant during column scale-up. 28. Any real-life protein feed even after clarification (filtration, 0.45 μm) may still contain components that can harm the column. A guard column is a small column packed from the same stationary phase as the separation column and inserted into the flow line just ahead of the separation column. Most of the damage is then done in the guard column, which is regularly replaced after a few cycles. 29. Instead of regeneration with pure Buffer B, it is possible to regenerate with a higher salt buffer (e.g., 2 M NaCl in Buffer A). 30. A conductivity detector is a good means to see when the high salt buffer has been completely displaced from the column during reequilibration.
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31. When increasing the flow rate, keep the pressure limit of the column and other system components (detector cell!) in mind. 32. The ability of a salt to enforce hydrophobic interaction is linked to its position in the Hofmeister series [29]. Sodium sulfate is a good alternative to ammonium sulfate. NaCl is a comparatively weak enforcer of hydrophobic interactions. 33. Column selection is not always straightforward in HIC as the hydrophobicity of a protein is difficult to predict. Very hydrophobic proteins need mildly hydrophobic stationary phases else the interaction becomes too strong (difficult elution) and vice versa. Binding strength will increase with ligands in the following order: ether
