Exploration of Ph-Gradient Ion-Exchange Chromatography for High-Resolution Protein Separations in Biotechnology and Proteomics

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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

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 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. J. NH3 Buffer/System pH 0 and applicability of pH-based IEC gradients for a wide range of proteins, Gradient: 0–100% B in 15 min Gradient: 0–100% B in 15 min Chromatogr., A 2008, 1194 (1), 22–29. 2 3456789 10 Flow Rate: 1.00 mL/min Flow Rate: 1.00 mL/min as well as the universality of the technique. Because of the flat nature of Detection: UV at 280 nm Detection: UV at 280 nm 28340 Buffer pH typically > pl 2. Farnan, D.; Moreno, G. T. Multi-Product High-Resolution Monoclonal Anion-Exchange general protein titration curves (typically from pH 6 to approximately pH 9) Anion-Exchange Resin Antibody Charge Variant Separations by pH Gradient Ion-Exchange Chromatography neutral proteins exhibit nearly zero net charge at a pH much higher than COO- their pI. The net charge of acidic and basic proteins approaches zero only Chromatography. Anal. Chem. 2009, 81 (21), 8846–8857. R NH 2 when pH is equal to pI. Therefore, the applicability of pH as an IEC design FIGURE 6. pH-gradient-based IEC of a monoclonal antibody – separation using a MAbPac SCX-10, 4 mm i.d. × 250 mm column. 3. Rea, J. C.; Moreno, G. T.; Lou, Y.; Farnan, D. Validation of a pH Anionic protein binds parameter is generally limited to acidic and basic proteins, or to determine Gradient-Based Ion-Exchange Chromatography Method for High- to positively charged an accurate pI. anion exchanger Column: MAbPac SCX-10, 4 mm i.d. × 250 mm Resolution Monoclonal Antibody Charge Variant Separations, Protein net charge vs pH Mobile Figure 4 shows some examples of pH-gradient-based anion-exchange 4 J. Pharm. Biomed. Anal. 2011, 54 (2), 317–323. chromatography (AEC) vs salt-gradient-based AEC, in which an Phase: A: 2.4 mM Tris + 1.5 mM imidazole 28336 + 11.6 mM piperazine, 4. Kaliszan, R.; Wiczling, P.; Markuszewski, M. J. pH Gradient High- attempt was made to keep gradients, elution windows, and gross peak titrated to pH 9.7 with HCl widths similar. Under these conditions, pH gradient-based AEC permited B: 2.4 mM Tris + 1.5 mM imidazole Performance Liquid Chromatography: Theory and Applications. J. facile separation of the three known isoforms of BSA resulting from +11.6 mM piperazine, Chromatogr., A 2004,1060, 165–175. titrated to pH 3.7 with HCl Instrumenta thiol-disulfide exchange. pH-based-gradient AEC was also found to be Gradient: 0–100% B in 25 min 5. Ahamed, T. et al., pH-Gradient Ion-Exchange Chromatography: An mAU HPLC experiments were carried out using a Thermo Scientific Dionex superior to salt-gradient-based AEC for albumin. Flow Rate: 1.00 mL/min Analytical Tool for Design and Optimization of Protein Separations. J. Detection: UV at 280 nm UltiMate 3000 Titanium system equipped with: Chromatogr., A 2007, 1164, 181–188. • SRD-3600 Solvent Rack with low-volume, chemically-inert degasser 6. Tsonev, L. I.; Hirsh, A. G. Theory and Applications of a Novel Ion • DGP-3600BM × 2 Biocompatible Dual-Gradient Micro Pump Exchange Chromatographic Technology Using Controlled pH 2 Exploration of pH-Gradient Ion-Exchange Chromatography• TCC-3000SD for Thermostatted High-Resolution Column Protein Compartment Separations in Biotechnology and Proteomics -0.5 Gradients for Separating Proteins on Anionic and Cationic Stationary • WPS-3000TBFC Thermostatted Biocompatible Autosampler with two Phases, J. Chromatogr., A 2008, 1200, 166–182. integrated switching valves 025710 12 15 17 20 22 25 • VWD-3400RS Variable Wavelength Detector with a 2.5 µL flow cell Minutes 28341 • PCM-3000 pH and Conductivity Monitor Enhancing Sample Throughput in Charge Variant Analysis Depending on the requirements set for the charge-variant analysis, the gain in analysis time may become more important than the loss of an

acceptable level of separation power. In this case, there are several All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. options which do not seriously affect the resolution. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. One can accelerate the current method by increasing the gradient slope, PO70013_E 02/12S or maintain the same gradient while utilizing a high-throughput (shorter) column. The example shown in Figure 7 illustrates a relatively small loss in resolution compared with trace B in Figure 5, even though the total analysis time was reduced more than fourfold. Method robustness was also unaffected by the reduction in analysis time. The lysine truncations are depicted as: LT1, no lysine; LT2, one lysine; LT3, two lysines. 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

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. J. NH3 Buffer/System pH 0 and applicability of pH-based IEC gradients for a wide range of proteins, Gradient: 0–100% B in 15 min Gradient: 0–100% B in 15 min Chromatogr., A 2008, 1194 (1), 22–29. 2 3456789 10 Flow Rate: 1.00 mL/min Flow Rate: 1.00 mL/min as well as the universality of the technique. Because of the flat nature of Detection: UV at 280 nm Detection: UV at 280 nm 28340 Buffer pH typically > pl 2. Farnan, D.; Moreno, G. T. Multi-Product High-Resolution Monoclonal Anion-Exchange general protein titration curves (typically from pH 6 to approximately pH 9) Anion-Exchange Resin Antibody Charge Variant Separations by pH Gradient Ion-Exchange Chromatography neutral proteins exhibit nearly zero net charge at a pH much higher than COO- their pI. The net charge of acidic and basic proteins approaches zero only Chromatography. Anal. Chem. 2009, 81 (21), 8846–8857. R NH 2 when pH is equal to pI. Therefore, the applicability of pH as an IEC design FIGURE 6. pH-gradient-based IEC of a monoclonal antibody – separation using a MAbPac SCX-10, 4 mm i.d. × 250 mm column. 3. Rea, J. C.; Moreno, G. T.; Lou, Y.; Farnan, D. Validation of a pH Anionic protein binds parameter is generally limited to acidic and basic proteins, or to determine Gradient-Based Ion-Exchange Chromatography Method for High- to positively charged an accurate pI. anion exchanger Column: MAbPac SCX-10, 4 mm i.d. × 250 mm Resolution Monoclonal Antibody Charge Variant Separations, Protein net charge vs pH Mobile Figure 4 shows some examples of pH-gradient-based anion-exchange 4 J. Pharm. Biomed. Anal. 2011, 54 (2), 317–323. chromatography (AEC) vs salt-gradient-based AEC, in which an Phase: A: 2.4 mM Tris + 1.5 mM imidazole + 11.6 mM piperazine, 28336 attempt was made to keep gradients, elution windows, and gross peak 4. Kaliszan, R.; Wiczling, P.; Markuszewski, M. J. pH Gradient High- titrated to pH 9.7 with HCl Performance Liquid Chromatography: Theory and Applications. J. Exploration of pH-Gradient Ion-Exchange Chromatographywidths similar. Under these conditions, pH gradient-based AEC permited B: 2.4 mM Tris + 1.5 mM imidazole facile separation of the three known isoforms of BSA resulting from +11.6 mM piperazine, Chromatogr., A 2004,1060, 165–175. titrated to pH 3.7 with HCl Instrumenta thiol-disulfide exchange. pH-based-gradient AEC was also found to be Gradient: 0–100% B in 25 min 5. Ahamed, T. et al., pH-Gradient Ion-Exchange Chromatography: An mAU High-Resolution Protein HPLCSeparations experiments were carried out using in a Thermo Biotechnology Scientific Dionex superior to salt-gradient-based and AECProteomics for albumin. Flow Rate: 1.00 mL/min Analytical Tool for Design and Optimization of Protein Separations. J. Detection: UV at 280 nm UltiMate 3000 Titanium system equipped with: Chromatogr., A 2007, 1164, 181–188. 1 2 • SRD-3600 Solvent Rack with low-volume,2 chemically-inert degasser 2 2 Gurmil Gendeh, Wim Decrop, Marie-Jeanne Olivo, Evert-Jan Sneekes, and Remco Swart 6. Tsonev, L. I.; Hirsh, A. G. Theory and Applications of a Novel Ion • DGP-3600BM × 2 Biocompatible Dual-Gradient Micro Pump Exchange Chromatographic Technology Using Controlled pH 1 2 Thermo Fisher Scientific, Sunnyvale,• TCC-3000SD CA, Thermostatted USA; Column Thermo Compartment Fisher Scientific, Amsterdam, The Netherlands-0.5 Gradients for Separating Proteins on Anionic and Cationic Stationary • WPS-3000TBFC Thermostatted Biocompatible Autosampler with two Phases, J. Chromatogr., A 2008, 1200, 166–182. integrated switching valves 025710 12 15 17 20 22 25 • VWD-3400RS Variable Wavelength Detector with a 2.5 µL flow cell Minutes 28341 • PCM-3000 pH and Conductivity Monitor Enhancing Sample Throughput in Charge Abstract FIGURE 2. The Thermo Scientific Dionex PCM-3000 is a new inert pH FIGURE 4. Comparison of pH-gradient-based AEC (left) and VariantFIGURE 7. Analysis 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). Depending on the requirements set for the charge-variant analysis, the for profiling the charge heterogeneity of biotherapeutic proteins, including quick response time. This unit includes a platform housing the pH gain in analysis time may becomeColumn: more importantMAbPac SCX-10, than the 4 mm loss i.d. o× f150 an 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 acceptable level of separation power. In this case, there are several 50 10 70 50 B: 20 mM MES pH 5.6 + 300 mM NaCl All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. ionic-strength-based ion-exchange separations are product specific UV-vis detector. Gradient: 0–100% B in 15 min This information is not intended to encourage use of these products in any manners that might infringe the options which do not seriouslyLT1 affect the resolution. and time consuming to develop. Although salt gradients are more Flow Rate: 1.00 mL/min intellectual property rights of others. One50 can accelerate the current Detection:method by increasingUV at 280 nm the gradient slope, LT2 PO70013_E 02/12S pH

commonly applied, the utilization of pH gradients can provide significant mAU mAU or maintain the same gradient while utilizing a high-throughput (shorter) LT3 advantages such as: 1) improved separation resolution; 2) lower salt (mS/cm)

Conductivity column. The example shown in Figure 7 illustrates a relatively small loss concentration in collected fractions; and 3) the possibility to correlate 0 0 in resolution compared with trace B in Figure 5, even though the total the protein isoelectric point (pI) data with elution profiles. -5 6.5 -10 -5 02 468101214 02 468101214 analysis time was reduced more than fourfold. Method robustness was Recently, the application of pH-gradient IEC has been described for the alsomAU unaffected by the reduction in analysis time. The lysine truncations 1 2,3 Ovalbumin separation of standard proteins and monoclonal antibodies. 35 10 30 50 are depicted as: LT1, no lysine; LT2, one lysine; LT3, two lysines. 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 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. J. NH3 Buffer/System pH 0 and applicability of pH-based IEC gradients for a wide range of proteins, Gradient: 0–100% B in 15 min Gradient: 0–100% B in 15 min Chromatogr., A 2008, 1194 (1), 22–29. 2 3456789 10 Flow Rate: 1.00 mL/min Flow Rate: 1.00 mL/min as well as the universality of the technique. Because of the flat nature of Detection: UV at 280 nm Detection: UV at 280 nm 28340 Buffer pH typically > pl 2. Farnan, D.; Moreno, G. T. Multi-Product High-Resolution Monoclonal Anion-Exchange general protein titration curves (typically from pH 6 to approximately pH 9) Anion-Exchange Resin Antibody Charge Variant Separations by pH Gradient Ion-Exchange Chromatography neutral proteins exhibit nearly zero net charge at a pH much higher than COO- their pI. The net charge of acidic and basic proteins approaches zero only Chromatography. Anal. Chem. 2009, 81 (21), 8846–8857. R NH2 FIGURE 6. pH-gradient-based IEC of a monoclonal antibody when pH is equal to pI. Therefore, the applicability of pH as an IEC design 3. Rea, J. C.; Moreno, G. T.; Lou, Y.; Farnan, D. Validation of a pH – Thermo Scientificseparation Poster usingNote• PN70013a MAbPac_e SCX-10,08/12S 43 mm i.d. × 250 mm column. Anionic protein binds parameter is generally limited to acidic and basic proteins, or to determine Gradient-Based Ion-Exchange Chromatography Method for High- to positively charged an accurate pI. anion exchanger Column: MAbPac SCX-10, 4 mm i.d. × 250 mm Resolution Monoclonal Antibody Charge Variant Separations, Protein net charge vs pH Mobile Figure 4 shows some examples of pH-gradient-based anion-exchange 4 J. Pharm. Biomed. Anal. 2011, 54 (2), 317–323. chromatography (AEC) vs salt-gradient-based AEC, in which an Phase: A: 2.4 mM Tris + 1.5 mM imidazole 28336 + 11.6 mM piperazine, 4. Kaliszan, R.; Wiczling, P.; Markuszewski, M. J. pH Gradient High- attempt was made to keep gradients, elution windows, and gross peak titrated to pH 9.7 with HCl widths similar. Under these conditions, pH gradient-based AEC permited B: 2.4 mM Tris + 1.5 mM imidazole Performance Liquid Chromatography: Theory and Applications. J. facile separation of the three known isoforms of BSA resulting from +11.6 mM piperazine, Chromatogr., A 2004,1060, 165–175. titrated to pH 3.7 with HCl Instrumenta thiol-disulfide exchange. pH-based-gradient AEC was also found to be Gradient: 0–100% B in 25 min 5. Ahamed, T. et al., pH-Gradient Ion-Exchange Chromatography: An mAU HPLC experiments were carried out using a Thermo Scientific Dionex superior to salt-gradient-based AEC for albumin. Flow Rate: 1.00 mL/min Analytical Tool for Design and Optimization of Protein Separations. J. Detection: UV at 280 nm UltiMate 3000 Titanium system equipped with: Chromatogr., A 2007, 1164, 181–188. • SRD-3600 Solvent Rack with low-volume, chemically-inert degasser 6. Tsonev, L. I.; Hirsh, A. G. Theory and Applications of a Novel Ion • DGP-3600BM × 2 Biocompatible Dual-Gradient Micro Pump Exchange Chromatographic Technology Using Controlled pH • TCC-3000SD Thermostatted Column Compartment -0.5 Gradients for Separating Proteins on Anionic and Cationic Stationary • WPS-3000TBFC Thermostatted Biocompatible Autosampler with two Phases, J. Chromatogr., A 2008, 1200, 166–182. integrated switching valves 025710 12 15 17 20 22 25 • VWD-3400RS Variable Wavelength Detector with a 2.5 µL flow cell Minutes 28341 • PCM-3000 pH and Conductivity Monitor Enhancing Sample Throughput in Charge Variant Analysis Depending on the requirements set for the charge-variant analysis, the gain in analysis time may become more important than the loss of an

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. J. NH3 Buffer/System pH 0 and applicability of pH-based IEC gradients for a wide range of proteins, Gradient: 0–100% B in 15 min Gradient: 0–100% B in 15 min Chromatogr., A 2008, 1194 (1), 22–29. 2 3456789 10 Flow Rate: 1.00 mL/min Flow Rate: 1.00 mL/min as well as the universality of the technique. Because of the flat nature of Detection: UV at 280 nm Detection: UV at 280 nm 28340 Buffer pH typically > pl 2. Farnan, D.; Moreno, G. T. Multi-Product High-Resolution Monoclonal Anion-Exchange general protein titration curves (typically from pH 6 to approximately pH 9) Anion-Exchange Resin Antibody Charge Variant Separations by pH Gradient Ion-Exchange Chromatography neutral proteins exhibit nearly zero net charge at a pH much higher than COO- their pI. The net charge of acidic and basic proteins approaches zero only Chromatography. Anal. Chem. 2009, 81 (21), 8846–8857. R NH 2 when pH is equal to pI. Therefore, the applicability of pH as an IEC design FIGURE 6. pH-gradient-based IEC of a monoclonal antibody – separation using a MAbPac SCX-10, 4 mm i.d. × 250 mm column. 3. Rea, J. C.; Moreno, G. T.; Lou, Y.; Farnan, D. Validation of a pH Anionic protein binds parameter is generally limited to acidic and basic proteins, or to determine Gradient-Based Ion-Exchange Chromatography Method for High- to positively charged an accurate pI. anion exchanger Column: MAbPac SCX-10, 4 mm i.d. × 250 mm Resolution Monoclonal Antibody Charge Variant Separations, Protein net charge vs pH Mobile Figure 4 shows some examples of pH-gradient-based anion-exchange 4 J. Pharm. Biomed. Anal. 2011, 54 (2), 317–323. chromatography (AEC) vs salt-gradient-based AEC, in which an Phase: A: 2.4 mM Tris + 1.5 mM imidazole 28336 + 11.6 mM piperazine, 4. Kaliszan, R.; Wiczling, P.; Markuszewski, M. J. pH Gradient High- attempt was made to keep gradients, elution windows, and gross peak titrated to pH 9.7 with HCl widths similar. Under these conditions, pH gradient-based AEC permited B: 2.4 mM Tris + 1.5 mM imidazole Performance Liquid Chromatography: Theory and Applications. J. facile separation of the three known isoforms of BSA resulting from +11.6 mM piperazine, Chromatogr., A 2004,1060, 165–175. titrated to pH 3.7 with HCl Instrumenta thiol-disulfide exchange. pH-based-gradient AEC was also found to be Gradient: 0–100% B in 25 min 5. Ahamed, T. et al., pH-Gradient Ion-Exchange Chromatography: An mAU HPLC experiments were carried out using a Thermo Scientific Dionex superior to salt-gradient-based AEC for albumin. Flow Rate: 1.00 mL/min Analytical Tool for Design and Optimization of Protein Separations. J. Detection: UV at 280 nm UltiMate 3000 Titanium system equipped with: Chromatogr., A 2007, 1164, 181–188. • SRD-3600 Solvent Rack with low-volume, chemically-inert degasser 6. Tsonev, L. I.; Hirsh, A. G. Theory and Applications of a Novel Ion • DGP-3600BM × 2 Biocompatible Dual-Gradient Micro Pump Exchange Chromatographic Technology Using Controlled pH • TCC-3000SD Thermostatted Column Compartment 4 Exploration of pH-Gradient Ion-Exchange Chromatography-0.5 for High-Resolution Protein Separations in Biotechnology and ProteomicsGradients for Separating Proteins on Anionic and Cationic Stationary • WPS-3000TBFC Thermostatted Biocompatible Autosampler with two Phases, J. Chromatogr., A 2008, 1200, 166–182. integrated switching valves 025710 12 15 17 20 22 25 • VWD-3400RS Variable Wavelength Detector with a 2.5 µL flow cell Minutes 28341 • PCM-3000 pH and Conductivity Monitor Enhancing Sample Throughput in Charge Variant Analysis Depending on the requirements set for the charge-variant analysis, the gain in analysis time may become more important than the loss of an

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. J. NH3 Buffer/System pH 0 and applicability of pH-based IEC gradients for a wide range of proteins, Gradient: 0–100% B in 15 min Gradient: 0–100% B in 15 min Chromatogr., A 2008, 1194 (1), 22–29. 2 3456789 10 Flow Rate: 1.00 mL/min Flow Rate: 1.00 mL/min as well as the universality of the technique. Because of the flat nature of Detection: UV at 280 nm Detection: UV at 280 nm 28340 Buffer pH typically > pl 2. Farnan, D.; Moreno, G. T. Multi-Product High-Resolution Monoclonal Anion-Exchange general protein titration curves (typically from pH 6 to approximately pH 9) Anion-Exchange Resin Antibody Charge Variant Separations by pH Gradient Ion-Exchange Chromatography neutral proteins exhibit nearly zero net charge at a pH much higher than COO- their pI. The net charge of acidic and basic proteins approaches zero only Chromatography. Anal. Chem. 2009, 81 (21), 8846–8857. R NH 2 when pH is equal to pI. Therefore, the applicability of pH as an IEC design FIGURE 6. pH-gradient-based IEC of a monoclonal antibody – separation using a MAbPac SCX-10, 4 mm i.d. × 250 mm column. 3. Rea, J. C.; Moreno, G. T.; Lou, Y.; Farnan, D. Validation of a pH Anionic protein binds parameter is generally limited to acidic and basic proteins, or to determine Gradient-Based Ion-Exchange Chromatography Method for High- to positively charged an accurate pI. anion exchanger Column: MAbPac SCX-10, 4 mm i.d. × 250 mm Resolution Monoclonal Antibody Charge Variant Separations, Protein net charge vs pH Mobile Figure 4 shows some examples of pH-gradient-based anion-exchange 4 J. Pharm. Biomed. Anal. 2011, 54 (2), 317–323. chromatography (AEC) vs salt-gradient-based AEC, in which an Phase: A: 2.4 mM Tris + 1.5 mM imidazole 28336 + 11.6 mM piperazine, 4. Kaliszan, R.; Wiczling, P.; Markuszewski, M. J. pH Gradient High- attempt was made to keep gradients, elution windows, and gross peak titrated to pH 9.7 with HCl widths similar. Under these conditions, pH gradient-based AEC permited B: 2.4 mM Tris + 1.5 mM imidazole Performance Liquid Chromatography: Theory and Applications. J. facile separation of the three known isoforms of BSA resulting from +11.6 mM piperazine, Chromatogr., A 2004,1060, 165–175. titrated to pH 3.7 with HCl Instrumenta thiol-disulfide exchange. pH-based-gradient AEC was also found to be Gradient: 0–100% B in 25 min 5. Ahamed, T. et al., pH-Gradient Ion-Exchange Chromatography: An mAU HPLC experiments were carried out using a Thermo Scientific Dionex superior to salt-gradient-based AEC for albumin. Flow Rate: 1.00 mL/min Analytical Tool for Design and Optimization of Protein Separations. J. Detection: UV at 280 nm UltiMate 3000 Titanium system equipped with: Chromatogr., A 2007, 1164, 181–188. • SRD-3600 Solvent Rack with low-volume, chemically-inert degasser 6. Tsonev, L. I.; Hirsh, A. G. Theory and Applications of a Novel Ion • DGP-3600BM × 2 Biocompatible Dual-Gradient Micro Pump Exchange Chromatographic Technology Using Controlled pH • TCC-3000SD Thermostatted Column Compartment -0.5 Gradients for Separating Proteins on Anionic and Cationic Stationary • WPS-3000TBFC Thermostatted Biocompatible Autosampler with two Phases, J. Chromatogr., A 2008, 1200, 166–182. integrated switching valves 025710 12 15 17 20 22 25 • VWD-3400RS Variable Wavelength Detector with a 2.5 µL flow cell Minutes 28341 • PCM-3000 pH and Conductivity Monitor Exploration of pH-Gradient Ion-Exchange Chromatography Enhancing Sample Throughput in Charge Variant Analysis High-Resolution Protein Separations in Biotechnology and Proteomics Depending on the requirements set for the charge-variant analysis, the gain in analysis time may become more important than the loss of an

acceptable level of separation power. In this case, there are several All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. 1 2 2 2 2 options which do not seriously affect the resolution. This information is not intended to encourage use of these products in any manners that might infringe the Gurmil Gendeh, Wim Decrop, Marie-Jeanne Olivo, Evert-Jan Sneekes, and Remco Swart intellectual property rights of others. 1 2 One can accelerate the current method by increasing the gradient slope, PO70013_E 02/12S Thermo Fisher Scientific, Sunnyvale, CA, USA; Thermo Fisher Scientific, Amsterdam, The Netherlandsor maintain the same gradient while utilizing a high-throughput (shorter) column. The example shown in Figure 7 illustrates a relatively small loss in resolution compared with trace B in Figure 5, even though the total analysis time was reduced more than fourfold. Method robustness was also unaffected by the reduction in analysis time. The lysine truncations are depicted as: LT1, no lysine; LT2, one lysine; LT3, two lysines.

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 Thermo Scientific Poster Note• PN70013_e 08/12S 5 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. J. NH3 Buffer/System pH 0 and applicability of pH-based IEC gradients for a wide range of proteins, Gradient: 0–100% B in 15 min Gradient: 0–100% B in 15 min Chromatogr., A 2008, 1194 (1), 22–29. 2 3456789 10 Flow Rate: 1.00 mL/min Flow Rate: 1.00 mL/min as well as the universality of the technique. Because of the flat nature of Detection: UV at 280 nm Detection: UV at 280 nm 28340 Buffer pH typically > pl 2. Farnan, D.; Moreno, G. T. Multi-Product High-Resolution Monoclonal Anion-Exchange general protein titration curves (typically from pH 6 to approximately pH 9) Anion-Exchange Resin Antibody Charge Variant Separations by pH Gradient Ion-Exchange Chromatography neutral proteins exhibit nearly zero net charge at a pH much higher than COO- their pI. The net charge of acidic and basic proteins approaches zero only Chromatography. Anal. Chem. 2009, 81 (21), 8846–8857. R NH 2 when pH is equal to pI. Therefore, the applicability of pH as an IEC design FIGURE 6. pH-gradient-based IEC of a monoclonal antibody – separation using a MAbPac SCX-10, 4 mm i.d. × 250 mm column. 3. Rea, J. C.; Moreno, G. T.; Lou, Y.; Farnan, D. Validation of a pH Anionic protein binds parameter is generally limited to acidic and basic proteins, or to determine Gradient-Based Ion-Exchange Chromatography Method for High- to positively charged an accurate pI. anion exchanger Column: MAbPac SCX-10, 4 mm i.d. × 250 mm Resolution Monoclonal Antibody Charge Variant Separations, Protein net charge vs pH Mobile Figure 4 shows some examples of pH-gradient-based anion-exchange 4 J. Pharm. Biomed. Anal. 2011, 54 (2), 317–323. chromatography (AEC) vs salt-gradient-based AEC, in which an Phase: A: 2.4 mM Tris + 1.5 mM imidazole 28336 + 11.6 mM piperazine, 4. Kaliszan, R.; Wiczling, P.; Markuszewski, M. J. pH Gradient High- attempt was made to keep gradients, elution windows, and gross peak titrated to pH 9.7 with HCl widths similar. Under these conditions, pH gradient-based AEC permited B: 2.4 mM Tris + 1.5 mM imidazole Performance Liquid Chromatography: Theory and Applications. J. facile separation of the three known isoforms of BSA resulting from +11.6 mM piperazine, Chromatogr., A 2004,1060, 165–175. titrated to pH 3.7 with HCl Instrumenta thiol-disulfide exchange. pH-based-gradient AEC was also found to be Gradient: 0–100% B in 25 min 5. Ahamed, T. et al., pH-Gradient Ion-Exchange Chromatography: An mAU HPLC experiments were carried out using a Thermo Scientific Dionex superior to salt-gradient-based AEC for albumin. Flow Rate: 1.00 mL/min Analytical Tool for Design and Optimization of Protein Separations. J. Detection: UV at 280 nm UltiMate 3000 Titanium system equipped with: Chromatogr., A 2007, 1164, 181–188. • SRD-3600 Solvent Rack with low-volume, chemically-inert degasser 6. Tsonev, L. I.; Hirsh, A. G. Theory and Applications of a Novel Ion • DGP-3600BM × 2 Biocompatible Dual-Gradient Micro Pump Exchange Chromatographic Technology Using Controlled pH • TCC-3000SD Thermostatted Column Compartment -0.5 Gradients for Separating Proteins on Anionic and Cationic Stationary • WPS-3000TBFC Thermostatted Biocompatible Autosampler with two Phases, J. Chromatogr., A 2008, 1200, 166–182. integrated switching valves 025710 12 15 17 20 22 25 • VWD-3400RS Variable Wavelength Detector with a 2.5 µL flow cell Minutes 28341 • PCM-3000 pH and Conductivity Monitor Enhancing Sample Throughput in Charge Variant Analysis Depending on the requirements set for the charge-variant analysis, the gain in analysis time may become more important than the loss of an acceptable level of separation power. In this case, there are several All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. options which do not seriously affect the resolution. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. One can accelerate the current method by increasing the gradient slope, PO70013_E 02/12S or maintain the same gradient while utilizing a high-throughput (shorter) column. The example shown in Figure 7 illustrates a relatively small loss in resolution compared with trace B in Figure 5, even though the total analysis time was reduced more than fourfold. Method robustness was also unaffected by the reduction in analysis time. The lysine truncations are depicted as: LT1, no lysine; LT2, one lysine; LT3, two lysines. 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

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. J. NH3 Buffer/System pH 0 and applicability of pH-based IEC gradients for a wide range of proteins, Gradient: 0–100% B in 15 min Gradient: 0–100% B in 15 min Chromatogr., A 2008, 1194 (1), 22–29. 2 3456789 10 Flow Rate: 1.00 mL/min Flow Rate: 1.00 mL/min as well as the universality of the technique. Because of the flat nature of Detection: UV at 280 nm Detection: UV at 280 nm 28340 Buffer pH typically > pl 2. Farnan, D.; Moreno, G. T. Multi-Product High-Resolution Monoclonal Anion-Exchange general protein titration curves (typically from pH 6 to approximately pH 9) Anion-Exchange Resin Antibody Charge Variant Separations by pH Gradient Ion-Exchange Chromatography neutral proteins exhibit nearly zero net charge at a pH much higher than COO- their pI. The net charge of acidic and basic proteins approaches zero only Chromatography. Anal. Chem. 2009, 81 (21), 8846–8857. R NH 2 when pH is equal to pI. Therefore, the applicability of pH as an IEC design FIGURE 6. pH-gradient-based IEC of a monoclonal antibody – separation using a MAbPac SCX-10, 4 mm i.d. × 250 mm column. 3. Rea, J. C.; Moreno, G. T.; Lou, Y.; Farnan, D. Validation of a pH Anionic protein binds parameter is generally limited to acidic and basic proteins, or to determine Gradient-Based Ion-Exchange Chromatography Method for High- to positively charged an accurate pI. anion exchanger Column: MAbPac SCX-10, 4 mm i.d. × 250 mm Resolution Monoclonal Antibody Charge Variant Separations, Protein net charge vs pH Mobile Figure 4 shows some examples of pH-gradient-based anion-exchange 4 J. Pharm. Biomed. Anal. 2011, 54 (2), 317–323. chromatography (AEC) vs salt-gradient-based AEC, in which an Phase: A: 2.4 mM Tris + 1.5 mM imidazole 28336 + 11.6 mM piperazine, 4. Kaliszan, R.; Wiczling, P.; Markuszewski, M. J. pH Gradient High- attempt was made to keep gradients, elution windows, and gross peak titrated to pH 9.7 with HCl widths similar. Under these conditions, pH gradient-based AEC permited B: 2.4 mM Tris + 1.5 mM imidazole Performance Liquid Chromatography: Theory and Applications. J. facile separation of the three known isoforms of BSA resulting from +11.6 mM piperazine, Chromatogr., A 2004,1060, 165–175. titrated to pH 3.7 with HCl Instrumenta thiol-disulfide exchange. pH-based-gradient AEC was also found to be Gradient: 0–100% B in 25 min 5. Ahamed, T. et al., pH-Gradient Ion-Exchange Chromatography: An mAU HPLC experiments were carried out using a Thermo Scientific Dionex superior to salt-gradient-based AEC for albumin. Flow Rate: 1.00 mL/min Analytical Tool for Design and Optimization of Protein Separations. J. Detection: UV at 280 nm UltiMate 3000 Titanium system equipped with: Chromatogr., A 2007, 1164, 181–188. • SRD-3600 Solvent Rack with low-volume, chemically-inert degasser 6. Tsonev, L. I.; Hirsh, A. G. Theory and Applications of a Novel Ion • DGP-3600BM × 2 Biocompatible Dual-Gradient Micro Pump Exchange Chromatographic Technology Using Controlled pH • TCC-3000SD Thermostatted Column Compartment -0.5 Gradients for Separating Proteins on Anionic and Cationic Stationary • WPS-3000TBFC Thermostatted Biocompatible Autosampler with two Phases, J. Chromatogr., A 2008, 1200, 166–182. integrated switching valves 025710 12 15 17 20 22 25 • VWD-3400RS Variable Wavelength Detector with a 2.5 µL flow cell Minutes 28341 • PCM-3000 pH and Conductivity Monitor Enhancing Sample Throughput in Charge Variant Analysis Depending on the requirements set for the charge-variant analysis, the gain in analysis time may become more important than the loss of an acceptable level of separation power. In this case, there are several All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. options which do not seriously affect the resolution. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. One can accelerate the current method by increasing the gradient slope, PO70013_E 02/12S or maintain the same gradient while utilizing a high-throughput (shorter) column. The example shown in Figure 7 illustrates a relatively small loss in resolution compared with trace B in Figure 5, even though the total analysis time was reduced more than fourfold. Method robustness was also unaffected by the reduction in analysis time. The lysine truncations are depicted as: LT1, no lysine; LT2, one lysine; LT3, two lysines.

6 Exploration of pH-Gradient Ion-Exchange Chromatography for High-Resolution Protein Separations in Biotechnology and Proteomics www.thermoscientific.com/dionex ©2012 Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products Thermo Scientific Dionex products are in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to designed, developed, and manufactured under an ISO 9001 Quality System. change. Not all products are available in all countries. Please consult your local sales representative for details.

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