Exploration of pH-Gradient Ion-Exchange Chromatography for High-Resolution Protein Separations in Biotechnology and Proteomics Gurmil Gendeh,1 Wim Decrop,2 Marie-Jeanne Olivo,2 Evert-Jan Sneekes,2 and Remco Swart2 1Thermo Fisher Scientific, Sunnyvale, CA, USA; 2Thermo Fisher Scientific, Amsterdam, The Netherlands Exploration of pH-Gradient Ion-Exchange Chromatography High-Resolution Protein Separations in Biotechnology and Proteomics Gurmil Gendeh,1 Wim Decrop,2 Marie-Jeanne Olivo,2 Evert-Jan Sneekes,2 and Remco Swart2 1Thermo Fisher Scientific, Sunnyvale, CA, USA;2 Thermo Fisher Scientific, Amsterdam, The Netherlands Abstract FIGURE 2. The Thermo Scientific Dionex PCM-3000 is a new inert pH FIGURE 4. Comparison of pH-gradient-based AEC (left) and FIGURE 7. Example of an accelerated salt-gradient-based IEC. Ion-exchange chromatography (IEC) is a versatile separation technique and conductivity monitoring system with low-volume flow cells and salt-gradient-based AEC (right). for profiling the charge heterogeneity of biotherapeutic proteins, including quick response time. This unit includes a platform housing the pH Column: MAbPac SCX-10, 4 mm i.d. × 150 mm ™ Mobile Phase: A: 20 mM MES pH 5.6 + 60 mM NaCl monoclonal antibodies. Despite good resolving power and robustness, and conductivity flow cell and can be mounted on any UltiMate 3000 BSA 50 10 70 50 B: 20 mM MES pH 5.6 + 300 mM NaCl ionic-strength-based ion-exchange separations are product specific UV-vis detector. Gradient: 0–100% B in 15 min LT1 and time consuming to develop. Although salt gradients are more Flow Rate: 1.00 mL/min 50 Detection: UV at 280 nm LT2 pH commonly applied, the utilization of pH gradients can provide significant mAU mAU LT3 advantages such as: 1) improved separation resolution; 2) lower salt (mS/cm) concentration in collected fractions; and 3) the possibility to correlate Conductivity 0 0 the protein isoelectric point (pI) data with elution profiles. -5 6.5 -10 -5 02 468101214 02 468101214 Recently, the application of pH-gradient IEC has been described for the mAU 1 2,3 Ovalbumin separation of standard proteins and monoclonal antibodies. 35 10 30 50 The work shown here describes the application of pH-gradient IEC as compared to salt-gradient IEC for the separation of proteins from mAU pH various sources. High-resolution separations of a monoclonal antibody mAU (mS/cm) and its isoforms were achieved using a new, nonporous, strong Conductivity cation-exchange resin. Results were compared to those obtained 0 0 -5 6.5 -5 -5 0 with salt-gradient IEC. Complex protein mixtures typically found 02 468101214 02 468101214 -2 Minutes Minutes 0 1 2 3 4 5 6 7 8 in proteomics were separated with pH-gradient IEC. Developed Minutes 28342 methodology was validated for pH profile shape and precision, pH Gradient Salt Gradient retention-time precision, peak capacity, and robustness towards Column: ProPac SAX-10, Column: ProPac SAX-10, 4 mm i.d. × 250 mm 4 mm i.d. × 250 mm sample solvent composition. Mobile Phase: A: 20 mM Piperazine Mobile Phase: A: 20 mM TRIS, pH 8.5 The speed of pH-gradient-based IEC can also be increased + 20 mM triethanolamine B: Same as A + 0.5 M NaCl considerably, as shown in Figure 8. A run with a total analysis time of + 20 mM bis-tris propane Gradient: 0–100% B in 15 min 60 min was reduced to 30 min by using a shorter (50 mm) MAbPac Principles + 20 mM N-methylpiperazine, Flow Rate: 1.00 mL/min pH = 3.7 (titrated with HCl) Detection: UV at 280 nm SCX-10 column, while maintaining a similar gradient. There are two general mechanisms on which proteins are retained B: 20 mM Piperazine and eluted from IEC columns (Figure 1). Use of either a continuous salt + 20 mM triethanolamine + 20 mM bis-tris propane FIGURE 8. Example of an accelerated pH-gradient-based IEC. (ionic-strength) gradient or a pH gradient result in a high degree of protein + 20 mM N-methylpiperazine, fractionation based on protein charge. pH = 9.7 (titrated with HCl) FIGURE 3. Salt-gradient-based IEC at different pH levels reveals Gradient: 0–100% B in 15 min Column: MAbPac SCX-10, 4 mm i.d. × 250 mm In salt-gradient-based IEC, the pH of the buffer system is fixed. In addition Flow Rate: 1.00 mL/min Mobile Phase: A: 2.4 mM Tris + 1.5 mM imidazole the importance of buffer pH selection for selectivity of the Detection: UV at 280 nm + 11.6 mM piperazine, titrated to pH 9.7 to choosing the appropriate pH of the starting buffer, its ionic strength is 28338 chromatographic method. with HCl kept low since the affinity of proteins for IEC resins decreases as ionic B: 2.4 mM Tris + 1.5 mM imidazole strength increases. The proteins are then eluted by increasing the ionic + 11.6 mM piperazine, titrated to pH 3.7 3 with HCl strength (salt concentration) of the buffer to increase the competition 30 Gradient: 0–100% B in 25 min pH 6.2 B C Retention Time vs pH IEC for Monoclonal Antibody Analysis A Flow Rate: 1.00 mL/min between the buffer ions and proteins for charged groups on the IEC resin. 16 As a result, the interaction between the IEC resin and proteins is reduced, mAU Salt-based cation-exchange chromatography is the gold standard for Detection: UV at 280 nm -5 14 causing the proteins to elute. C charge variant analysis of monoclonal antibodies (MAbs). The Thermo 30 12 Scientific ProPac WCX-10 and Thermo Scientific MAbPac SCX-10 are pH 7.0 B C B mAU In pH-gradient-based IEC, the pH of the starting buffer is maintained at A 10 two high-performance, industry-leading, charge variant analysis columns, mAU A a constant level to ensure the proteins obtain the opposite charge of the 8 ™ -5 featuring unique selectivity and high resolving power. The MAbPac stationary phase and bind to it. The proteins are eluted by changing the Minutes 6 ™ 45 SCX-10 column is complimentary to the ProPac WCX-10 column for pH 7.6 buffer pH so the proteins transition to a net zero charge (ultimately the A + B 4 monoclonal antibody variant analysis. The MAbPac SCX-10 column C same charge as the resin) and elute from the column. One of the benefits 0 mAU 2 offers alternative selectivity and provides higher resolution and efficiency of pH-gradient-based IEC is that the salt concentration can be kept low, -5 0 for variant analysis of most monoclonal antibody samples than the -0.5 yielding less buffer interferences in, for example, on-line or off-line 40 0246810 12 15 B 6 788.5 ProPac WCX-10 column (see Figure 5). Figure 6 shows an analytical pH 8.2 Minutes 28343 two-dimensional LC (2D-LC). pH method utilizing a pH gradient. mAU A C High pI proteins are generally separated on cation-exchange columns -5 running a pH-based gradient from low to high pH, and vice versa for 0 2 4 6 8 10 12 14 FIGURE 5. Typical high-resolution, salt-gradient-based IEC Conclusions low pI proteins. Minutes chromatograms for separations using A) ProPac WCX-10, • pH-gradient-based IEC can be a very good alternative to salt- Column: Thermo Scientific Detection: UV at 280 nm 4 mm i.d. × 250 mm (left) and B) MAbPac SCX-10, 4 mm i.d. × 250 mm ProPac SCX-10, Peaks: A: α-Chymotrypsinogen (pI = 8.5) gradient-based IEC. FIGURE 1. The protein isoelectric point determines the buffer 4 mm i.d. × 250 mm B: Ribonuclease A (pI = 9.45) (right) columns. system and column selection. The scheme applies to both Mobile Phase: A: 25 mM Phosphate C: Cytochrome C (pI = 10.2) • Good resolution was found for pH-gradient-based separations with both B: Same as A + 0.5 M NaCl long and short SCX columns. salt-gradient-based IEC (one vertical line on the pH axis) as well Gradient: 0–50% B in 15 min AB8 as pH-gradient-based IEC (along the protein net charge line). Flow Rate: 1.00 mL/min 5 • One of the benefits of pH-gradient-based IEC is that the salt 28337 concentration can be kept low, yielding less buffer interferences (e.g., mAU mAU Cationic protein binds on-line or off-line two-dimensional LC [2D-LC]). Isoelectric to negatively charged • pH-gradient IEC is promising for high throughput and fast screening cation exchanger Point (pl) 0 - High-Resolution, pH-Based IEC of Intact Proteins COO of proteins and antibodies. + R NH+ -2 -1 3 Using pH as a foundation for separation is not new, as it is widely applied Cation-Exchange 0 10 20 30 40 50 0 10 20 30 40 50 Resin in the bioseparation field (e.g., electrophoresis). However, over the last Minutes Minutes Buffer pH typically < pl few years, pH-gradient-based IEC has emerged as a core analytical References Cation-Exchange Column: ProPac WCX-10, 4 mm i.d. × 250 mm Column: MAbPac SCX-10, 4 mm i.d. × 250 mm Chromatography method. Several research groups (e.g., Kaliszan, R. et al.; Ahamed, T. et Mobile Phase: A: 20 mM MES pH 5.6 + 60 mM NaCl Mobile Phase: A: 20 mM MES pH 5.6 + 60 mM NaCl 1. Ahamed, T. et al., Selection of pH-Related Parameters in Ion- COOH 2–6 R + al.; Tsonev, L. I. et al.; Farnan, D. et al. ) have demonstrated the power B: 20 mM MES pH 5.6 + 300 mM NaCl B: 20 mM MES pH 5.6 + 300 mM NaCl Exchange Chromatography Using pH-Gradient Operations.