596 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS Table 13.3 Initial Conditions for RPC Method Development Values for Different Samples Condition Peptides Proteins Sample 1<M < 5kDa 5< M < 20 kDa M > 20 kDa Sample treatment prior to injection None Add 8 M urea, store for 30 min Add 8M urea, store for 30 min Column a 150 × 4.6-mm, type-B C 18 (8–12 nm pore-diameter), 3-μm particles 150 × 4.6-mm, type-B C 18 (12–30 nm pore-diameter), 3-μm particles 50 × 4.6-mm, type-B C 4 (≥ 30 nm pore-diameter), 3-μm particles Solvent A 0.1% TFA—water 0.1% TFA—water 0.1% TFA—water Solvent B 0.10% TFA—ACN 0.10% TFA—ACN 0.10% TFA—ACN Gradient range 0–60% B 5–100% B 5–100% B Temperature 30–35 ◦ C30–35 ◦ C b 30–35 ◦ C b Flow rate (mL/min) 2.0 1.0 0.5 Gradient time (min) 25 50 50 k ∗ 211 %B/min 2.4 1.2 1.2 Value of S assumed 25 40 70 a Columns should be stable at low pH and temperatures ≤ 60 ◦ C; other column lengths, diameters and par- ticle sizes can be used, in which case gradient time and flow rate should be adjusted to maintain similar values of k ∗ with acceptable pressure drop. The choice of ligand length (C 8 ,C 18 )islesscritical. b Higher temperatures (e.g., 60–80 ◦ C) can be desirable for some protein samples, especially those with M > 20 kDa; column stability for these conditions should be verified before the use > 50 ◦ Cand pH < 2.5. Figure 13.10, the initial four runs a–d can be used to predict the best combination of temperature and gradient time for optimal resolution (Fig. 13.10e). Once acceptable peak spacing is achieved, the gradient range can be trimmed to shorten overall separation time. For example, the gradient can be initiated at a %B-value just prior to elution of the first peak, and terminated at the %B-value just after elution of the last peak (Fig. 13.10f ). If no combination of gradient time and temperature yields acceptable reso- lution, the next step could be a change in the column or the composition of the A- or B-solvent; for example, an increase in TFA concentration, a change in pH, or the substitution of isopropanol for acetonitrile as B-solvent. After one or more of the latter changes in conditions, the four-run change in both gradient time and temperature (as in Fig. 13.10a–d) should be repeated, using the new conditions for other variables. Finally, segmented gradients can be used to address particular separation problems. In the case of strongly adsorbed contaminants that must be removed from the column prior to the next sample injection, a final, steep gradient to 100% B can be used to clean the column. In the case of complex samples with clusters of poorly 13.4 SEPARATION OF PEPTIDES AND PROTEINS 597 resolved components, a segment with a shallow gradient ramp can be inserted to improve their separation. This strategy is of limited value for small molecules; it is more likely to be successful for peptides, and especially for proteins [36]. 13.4.2 Ion-Exchange Chromatography (IEC) and Related Techniques Ion-exchange chromatography (IEC) can be used for analytical separations of peptides and proteins, but it is more frequently employed for the isolation and purification of proteins from laboratory to process scale [37]. The most important advantages of IEC for protein isolation include (1) the tendency of proteins to maintain their native conformation and biological activity during separation, (2) the relatively high binding capacity of IEC packings, and (3) high mass recov- eries. Features (1) and (2) are favored by the use of mobile phases of moderate ionic strength and near-physiological pH. The most important feature of IEC for analytical applications is its unique selectivity relative to other modes of col- umn chromatography. Three other chromatographic techniques (chromatofocusing, hydroxyapaptite chromatography, and immobilized-metal affinity chromatography; Sections 13.4.2.3–13.4.2.5) are related to IEC in that they also rely on ionic interactions between the column and sample. Ion exchange is based on the reversible electrostatic interaction of charged groups on the packing with oppositely charged groups on the polypeptide (Section 7.4.1). The retention of a peptide or protein molecule P occurs as a result of the displacement of mobile-phase counterions X + by P +z (or X − by P −z ). (cation exchange) P +z (m) + z(R − )X + (s) ⇔ (R − ) z P +z (s) + zX + (m) (13.4) (anion exchange) P −z (m) + z(R + )X − (s) ⇔ (R + ) z P −z (s) + zX − (m) (13.5) Here R − or R + refers to a charged group (ligand) in the stationary phase, z is the charge on the protein molecule P +z or P −z ,and(m)or(s) refers to a molecule in the mobile or stationary phase, respectively. A monovalent counter-ion X + or X − is assumed in Equations (13.4) and (13.5). In cation-exchange chromatogra- phy, an anionic ligand (R − ) associates with cationic sites on the polypeptide. In anion-exchange chromatography, a positively charge ligand (R + ) binds to anionic groups on the polypeptide. Sample retention can be varied by altering the charge on the solute or—in some cases—the column ligand (Section 7.5.4) via a change in mobile-phase pH. A more common elution strategy is to vary the concentration of X + or X − in the mobile phase, as discussed in Section 7.4.1, or to use gradient elution where the concentration of X + or X − increases during the gradient (salt gradient). For reasons discussed below, the apparent charge ±z on the protein in Equations (13.4) and (13.5) can differ from the net charge. Charged groups at the protein amino and carboxyl termini (as well as on amino-acid side-chains) strongly affect IEC retention. These groups have pK a values between 2 and 13 (Table 13.4 and Fig. 13.1), so retention will be strongly dependent on mobile-phase pH. Note that the local environment of a charged amino-acid residue in a protein (i.e., surrounding mobile phase, and adjacent amino-acid groups within the molecule) can shift its apparent pK a from the nominal value for the free amino acid. Charged post-translational modifications such as sialic acid, phosphate, and sulfate groups can also contribute to ionic retention. 598 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS Table 13.4 pK a Values for Charged Amino Acids Residue pK a in Amino Acid pK a in Protein Terminal amino 8.8–10.8 6.8–7.9 Arginyl 12.5 ≥12 Histidyl 6.0 6.4–7.4 Lysyl 10.8 5.9–10.4 Terminal carboxyl 1.8–2.6 3.5–4.3 Aspartyl 3.9 4.0–7.3 Glutamyl 4.3 4.0–7.3 Source: Reprinted from [37] with permission from Validated Biosystems. The net charge ±z on a protein will depend on mobile-phase pH. At the pH where the sum of positive and negative charges are equal (the isoelectric point, or pI), no net IEC retention is expected. At pH values below its pI, a protein will have a net positive charge and should bind to a cation exchanger. At pH values above its pI, the protein will possess a net negative charge and should bind to an anion exchanger (Fig. 13.15a). This simple model can serve as a guide for selecting a column and mobile-phase pH, but in practice, a protein may exhibit anomalous binding behavior at or near its isoelectric point (Fig. 13.15b). The reason is that the charge on a protein may not be homogeneously distributed across its surface but instead clustered into different regions (contact areas) on the molecule (Section 13.3.2). As a result regions of excess charge can appear at different parts of the molecule, and these regions can interact with the column more or less independently of each other. Anomalous binding behavior can include binding at the isoelectric point, binding to an anion exchanger below the protein pI, or binding to a cation exchanger above the pI. Similarly a protein may fail to bind to an anion exchanger above its pI or to a cation exchanger below its pI. For example, β glucosidase (pI = 7.3) binds at pH-7.3 on an anion exchanger but fails to bind to a cation exchanger until the mobile-phase pH is two units below its pI (Fig. 13.15b). Chymotrypsin, with a pI of 9, binds at pH-9 on both an anion and a cation exchanger more than (Fig. 13.15c). As a guideline, anion-exchange separations are often carried out at 1 to 1.5 pH units above a protein’s pI, and cation-exchange separations at 1 to 1.5 pH units below the pI. Solubility and stability properties of the protein(s) of interest can limit the allowable ionic conditions for the separation. Virtually all protein purification schemes used in the biopharmaceutical industry contain one or multiple anion- and/or cation-exchange steps. Since only a limited number of charged residues on the protein surface may interact with the stationary phase, small differences in the nature and positions of these charged residues can profoundly affect selectivity in ion-exchange chromatog- raphy [14]. In addition amino-acid substitutions within the interior of the protein may alter its conformation and affect ion-exchange selectivity indirectly by changing the positions of charged groups on the protein surface. 13.4 SEPARATION OF PEPTIDES AND PROTEINS 599 (a) (b) (c) 20 10 0 2468 4 6 8 1010 2 pI pI t R (min) pH cation exchange anion exchange +z −z t R t R pI pH Cation exchange Anion exchange Protein charge z, retention time t R β−glucosidase chymotrypsinogen Figure 13.15 Protein retention on ion exchangers as a function of pH. Ideal behavior (a); actual behavior of β-glucosidase (b) and chymotrypsinogen (c). Adapted from [38]. 13.4.2.1 Column Selection Column-selection criteria include: • particle size and pore diameter • support composition • ligand type • ligand density Particle size and pore diameter considerations are the same as described in Sections 13.3.1.1 and 13.3.1.2 for RPC. Support Composition. The first supports for high-performance IEC were silica based, for the same reasons that silica was chosen for other modes of HPLC. However, early silica packings were unstable under preferred ion-exchange condi- tions (physiological pH, moderate salt concentration) and were gradually replaced by polymeric packings based on polystyrene-divinyl benzene or polymethacrylate. Although modern silica-based packings exhibit improved stability at neutral to 600 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS alkaline pH, many labs continue to use polymer-based columns. For process chro- matography, large-particle supports composed of semi-rigid gels such as cross-linked dextran, agarose, or polyacrylamide are preferred for their lower cost, and because they can withstand highly alkaline cleaning steps for the removal of endotoxins and other biological contaminants. Ligand Type and Density. Within the respective categories of cation and anion exchange, IEC packings can be further divided into ‘‘strong’’ or ‘‘weak’’—depending on the pK a of the stationary-phase ionic ligand. Consequently the charge on the column and its binding capacity can vary with mobile-phase pH (Fig. 13.16). Strong ion-exchangers have pK a values outside the normal pH-operating range of the column, and are therefore fully ionized—regardless of mobile-phase pH; see Table 13.5 for some common examples of IEC column ligands. Ionic groups in strong ion-exchangers include –SO 3 − for cation exchange and –N(CH 3 ) 3 + for anion exchange. Weak ion-exchangers have pK a values within the operating range of the column, so their ion-exchange capacity varies with mobile-phase pH. Examples of 0 2468101214 02468101214 Strong CEX Strong AEX Exchange Capacity Weak CEX Weak AEX Exchange Capacity (a) (b) pH p H Figure 13.16 Capacities of ion-exchange groups. (a) Strong ion exchangers; (b)weakion exchangers. Adapted from [39]. 13.4 SEPARATION OF PEPTIDES AND PROTEINS 601 Table 13.5 Strong and Weak Ion-Exchange Ligands Anion Exchange (AEX) Cation Exchange (CEX) Weak Weak DEAE (diethylaminoethyl) –O–CH 2 –CH 2 –N + H(CH 2 CH 3 ) 2 CM (Carboxymethyl) –O–CH 2 –COO − PEI (polyethyleneimine) (–NHCH 2 CH 2 ) n –N(CH 2 CH 2 –) n . | CH 2 CH 2 NH 2 Strong Strong Q (quaternary ammonium) –CHOH–CH 2 –N + (CH 3 ) 3 S (sulfonate) –CH 2 –CH 2 –CH 2 –SO − 3 weak IEC groups include –N(C 2 H 5 ) 2 H + for weak anion exchange and –COO − for weak cation exchange. Weak anion-exchange columns of polyethyleneimine consist of a dense polymeric coating onto a silica support, yielding a column with high capacity and good stability under alkaline conditions. Strong ion-exchangers are often preferred, as their exchange capacity is independent of mobile-phase pH and their behavior is more predictable. The binding capacity of ion-exchangers depends on the surface area of the support and its charge density (μmoles/m 2 ). Typical ion-exchange capacities (i.e., for maximum uptake of sample by the column) for large-pore silica or polymer-based columns are in the range of 30 to 120 mg protein per milliliter of packing. The linker group that joins the ion-exchange group to the support can con- tribute to the chromatographic properties of the column. For example, hydrophobic groups in the linker may participate in hydrophobic (reversed-phase) interactions with the solute. Such interactions can account for differences in column selectivity among different vendors who use the same ion-exchange functionality. Tentacle IEC stationary phases have a flexible hydrophilic linker (the ‘‘tentacle’’) that connects the charged group to the support [40]. These columns improve access of the protein to the charged group of the packing, thus enhancing binding capacity. In addition tentacle columns may exhibit reduced nonspecific interaction, improved binding kinetics, and reduced protein denaturation. 13.4.2.2 Mobile-Phase Selection As noted above, control of retention (solvent strength) is usually achieved by varying the concentration of a displacing salt (counter-ion), rather than by changes in mobile-phase pH. Conditions that affect selectivity include: • column (Section 13.4.2.1) • mobile-phase buffer • counter-ion salt type (as in Fig. 13.17) 602 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS 01020 30 010 1 2 3 + 4 5 1 2 3 4 5 (a)(b) NaCl Na 2 SO 4 ( min )( min ) Figure 13.17 Effect of salt type on anion exchange separation of five proteins. Conditions: 50 × 4-mm Shim-pack WAX-2 column (Shimadzu); 0–0.5M of indicated salt in 20 min; pH-8 phosphate buffer; 1 mL/min. Adapted from [41]. • gradient steepness • organic B-solvent (if used) • other mobile-phase additives (especially surfactants) • temperature See also the discussion of Section 7.5. Mobile-Phase Buffer. Achieving the desired retention and selectivity requires a careful selection and control of the mobile-phase pH. For a cation-exchange column, a mobile-phase pH near 6 is a good starting point, while a mobile-phase pH of 8 is appropriate for an anion exchanger. For good buffering capacity, the buffering agent should have a pK a value within roughly 1.0 units of the target pH (Section 7.2.1.1), and a concentration of 0.02 to 0.1 M. Common buffers used for IEC are listed in Table 7.1. Note that some of these buffers absorb strongly at shorter UV wavelengths, especially if higher concentrations are used. Counter-Ion. The most common elution strategy in IEC is the use of a gradient of increasing concentration of the counter-ion. The relative strength of different counter-ions follows their ranking in the Hofmeister series [37, 42]; see Table 13.6 or a similar series in Section 7.5.2. However, gradients that involve an increase in NaCl are most often used for both anion and cation exchange. Note that chloride is corrosive for stainless steel at low pH (<5) and should be removed from the HPLC system after use. However, special-purpose HPLC systems have been designed that enable the use of chloride under acidic conditions. Organic Solvents and Surfactants. Organic solvents (e.g 1–10% methanol, propanol, or acetonitrile) can be added to the mobile phase to suppress hydrophobic 13.4 SEPARATION OF PEPTIDES AND PROTEINS 603 Table 13.6 Hofmeister Series of Lyotropic and Chaotropic Ions [36] Increasing lyotropic (salting out) effect SCN − (least) < ClO 4 − < NO 3 − < Br − < Cl − < COO − < SO 4 2− < PO 4 3− (most) Increasing chaotropic (salting in) effect Ba 2+ (most) > Ca 2+ > Mg 2+ > Li + > Cs + > Na + > K + > Rb + > NH 4 + (least) Source: Data from [36]. interactions with the support or linker groups, and to decrease peak broadening or tailing (addition of as much as 50% organic solvent may be required in some cases, as in the example of Fig. 11.15 of [43]). Nonionic surfactants can also be used for the same reasons. Either of these mobile-phase additives can also maintain the solubility of very hydrophobic solutes such as membrane proteins. Ionic surfactants can not be used in ion-exchange chromatography. 13.4.2.3 Chromatofocusing Chromatofocusing is a specialized form of IEC in which proteins are eluted from the column with a pH gradient [44–49]. Chromatofocusing is unique in that the pH gradient is formed within the column, by means of a single mobile phase that is a complex mixture of different buffering species. Although chromatofocusing can be performed with cation- or anion-exchangers, commercially available products are limited to anion exchange [48]. At the start of separation, proteins are retained by the anion exchanger, which has been pre-equilibrated at high pH for maximum retention of the sample. Then a low-pH buffer mixture is used as mobile phase, which, upon moving through the column, progressively titrates the charge on the column so that pH increases along the column, from inlet to outlet. Proteins migrate down the column in response to the changing pH and elute at or near their isoelectric points—a pH at which they can no longer bind to the exchanger. Elution is in order of descending protein pI values. Chromatofocusing is characterized by very high capacity, so it is useful for preparative separations. The technique is also capable of very high resolution, by virtue of focusing effects that generate sharp peaks 0.04 to 0.05 pH units in width. As is the case for conventional IEC (Figs. 13.15b,c), a protein can elute from a chromatofocusing column at a pH that is significantly different from its pI. Successful and reproducible chromatofocusing separations depend on the use of buffers that contain multiple species, whose pK a values span the range of the pH gradient, and that can achieve effective buffering across this range. Commercial chromatofocusing buffers are composed of a mixture of ampholytes (substances that may act as either an acid or a base). Alternatively, a combination of biological buffers such as Good’s buffers [50] can be used. The ionic strength of the elution buffer must be kept low, in order to minimize salt-mediated elution (displacement of proteins by counter-ions). The improved resolution (or faster separation) of proteins whose pI values fall within a narrow range of values can be achieved by narrowing the pH range of the ampholyte or buffer blend (similar to a decrease in φ in 604 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS gradient elution). Strong ion-exchange columns are preferred for chromatofocusing, since they are fully ionized—regardless of pH. One shortcoming of chromatofocusing is the reduced solubility of proteins at their isoelectric point, a limitation which is exacerbated by the low ionic strength of the elution buffer. Protein solubility can be enhanced by an increase in salt concentration, but this will increase mobile-phase strength and compromise the separation. A preferred strategy for dealing with protein precipitation is the addition of zwitterions to the elution buffer. Additives such as taurine, glycine, and betaine promote protein solubility and can be used in concentrations up to 2M without affecting the ionic strength of the buffer. The addition of urea at concentrations of 1 to 2M also helps solubilize proteins; nonionic and zwitterionic surfactants may be used as well. Note, however, the tendency of urea to decompose to carbamates, which can covalently modify a protein. Chromatofocusing is able to resolve isoforms of proteins that have different charge states, for example, post-translationally modified proteins that differ in the number of sialic acids or phosphate groups. The resolution of isoforms can be a limitation, if the goal is protein purification. The target protein is then resolved into multiple peaks, which dilutes the target protein and increases the risk of co-elution with sample contaminants. On the other hand, this characteristic of chromatofocusing can be an advantage, if only the characterization of isoforms is desired. 13.4.2.4 Hydroxyapatite Chromatography This technique is frequently used in process chromatography for protein purification and the removal of contaminants [37]. Hydroxyapatite (HA) is a crystalline material composed of Ca 10 (PO 4 ) 6 (OH) 2 that serves both as the support and the stationary phase [51]. The multifunctional surface consists of positively charged pairs of calcium ions (C-sites) and clusters of six anionic oxygen atoms associated with triplets of phosphate ions (P-sites). The C- and P-sites and hydroxyls are distributed in a fixed pattern on the crystal surface [51–53], as illustrated in Figure 13.18. Early preparations of HA were unstable, but modern HA materials are sintered at high temperature to form ceramic hydroxyapatite (CHT), which is stable under chromatographic conditions. Columns packed with either 5- or 10-μm CHT particles are available for both analytical and preparative applications. Protein interactions with CHT are complex (Fig. 13.18). Electrostatic inter- actions include attraction of protonated amino groups by P-sites and repulsion by C-sites (Fig. 13.18a). Similarly ionized carboxyl groups are attracted by C-sites and repelled by P-sites (Fig. 13.18b). Although the initial attraction of carboxyls to C-sites is electrostatic, the actual binding involves formation of much stronger coordination complexes between C-sites and clusters of protein carboxyl-groups [37]. Protein phosphate-groups bind C-sites even more strongly than protein carboxyl-groups. The selectivity of CHT for basic proteins is distinct from that of conventional cation exchange, due to the repulsion of amines by C-sites. Binding of weakly basic proteins can be enhanced by the addition of a low concentration of phosphate, which suppresses C-site repulsion of amines but does not block their interaction with P-sites [54]. Basic proteins can be eluted by gradients of sodium chloride or phosphate; a final salt concentration as high as 0.5M may be required. Although the 13.4 SEPARATION OF PEPTIDES AND PROTEINS 605 OH OH OH Ca +)) Ca +)) PO 4 = PO 4 = PO 4 = ((+ H 2 N ((+ H 2 N + H 2 N + H 2 N (a) (b) COO − COO − COO −)) COO −)) (( PO 4 = PO 4 = (( PO 4 = + Ca + Ca HO HO HO Protein Protein CHT CHT C-sites P-sites Figure 13.18 Binding to ceramic hydroxyapatite (CHT) of a basic protein (a) and an acidic protein (b). Double parenthesis indicate repulsion, dotted lines indicate ionic bonds, and trian- gular linkages indicate coordination bonds. Adapted from [37]. binding of basic proteins increases at lower pH, CHT is unstable below pH 5. Acidic proteins cannot be eluted with sodium chloride—even at concentrations > 0.3M; their elution requires the use of phosphate, citrate, or fluoride. This characteristic of CHT permits separation of basic proteins with an initial NaCl gradient, followed by elution of acidic proteins with a phosphate gradient. CHT typically provides excellent recovery of protein mass and biological activity; it is used for protein purification from laboratory to process scale. The unique selectivity of CHT can enable the resolution of closely related species such as protein variants and glycoforms. It is used in the biopharmaceutical industry for the purification of antibodies and removal of contaminants such as endotoxins, nucleic acids, and viruses. The stability of CHT toward concentrated base, organic solvents, and chaotropes enables aggressive cleaning regimes to be applied after use. 13.4.2.5 Immobilized-Metal Affinity Chromatography (IMAC) This separation mode, also known as metal-interaction chromatography (MIC), is based on the differential interaction of proteins with a metal ion [55–57]. The metal ion is immobilized by chelating groups that are attached to the support via a linker; see the example of Figure 13.19, which includes the various steps in its use. Several . selectivity relative to other modes of col- umn chromatography. Three other chromatographic techniques (chromatofocusing, hydroxyapaptite chromatography, and immobilized-metal affinity chromatography; Sections. point, binding to an anion exchanger below the protein pI, or binding to a cation exchanger above the pI. Similarly a protein may fail to bind to an anion exchanger above its pI or to a cation exchanger. kDa Sample treatment prior to injection None Add 8 M urea, store for 30 min Add 8M urea, store for 30 min Column a 150 × 4.6-mm, type-B C 18 (8–12 nm pore-diameter), 3-μm particles 150 × 4.6-mm,