High-Throughput, High-Resolution Monoclonal Antibody Analysis with Small Particle Size HPLC Columns

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1 High-Throughput, High-Resolution Monoclonal ntibody nalysis with Small Particle Size HPLC Columns Srinivasa Rao, Yuanxue Hou, Hongmin Zhang, Yury groskin, and Chris Pohl Thermo Fisher Scientific, Sunnyvale, C pplication Note 008 Key Words MbPac SCX-0, ProPac WCX-0, high-throughput analysis, lysine truncation, mb separation, mb analysis, mb heterogeneity bstract High-throughput applications of the Thermo Scientific MbPac SCX-0 column are described, showing acidic and basic variant analysis, including mb lysine truncation variant characterization. The ruggedness of the μm small particle phase column is also demonstrated. Introduction Monoclonal antibodies (mbs) can undergo a number of different chemical modifications including oxidation, deamidation, and C-terminal lysine truncation during the manufacturing process [ ]. Manufacturing and subsequent quality control and stability assessment of mbs can be very challenging tasks. The increasing focus on the development of mbs in the pharmaceutical industry is driving a growing demand for improved high-resolution stationary phases for characterization of mbs. Higher throughput is required for quality control due to an increasing sample load in many labs. The MbPac SCX-0 column is a strong acid cationexchanger that is complementary to the widely used Thermo Scientific ProPac WCX-0 column. It provides high resolution and alternative selectivity for various proteins and mbs. oth ProPac WCX-0 and MbPac SCX-0 stationary phases are based on a nonporous highly cross-linked vinylaromatic type of polymeric media with a proprietary hydrophilic coating. The ProPac WCX-0 column is available in only a 0 µm particle size; however, the MbPac SCX-0 is available in 0, 5, and μm particle sizes. MbPac SCX-0 μm and 5 μm small particle columns are available in different formats to suit a variety of application needs. The 50 mm and 50 mm length columns are designed for high-resolution separations, and shorter 50 mm columns are used when high throughput is desired. This application note shows the selectivity differences between ProPac WCX-0 and MbPac SCX-0 phases for protein separation. pplications using smaller particle columns for high-throughput, high-resolution mb separations are also described. The ruggedness of small particle phase columns is presented.

2 Experimental Details Sample Handling Equipment Mb separations were performed on an inert Thermo Scientific Dionex UltiMate 000 Titanium system that was equipped with the following: DGP-00 M iocompatible dual gradient pump SRD-00 Solvent rack with six degasser channels WPS-000TFC nalytical thermostatted autosampler VWD-400 UV Detector equipped with micro flow cell TCC-000SD Thermostatted column compartment Columns Part Number ProPac WCX-0, 0 μm, 4 50 mm MbPac SCX-0, 0 μm, 4 50 mm 0745 MbPac SCX-0, 5 μm, 4 50 mm 0785 MbPac SCX-0, 5 μm, 4 50 mm MbPac SCX-0, 5 μm, 4 50 mm MbPac SCX-0, μm, 4 50 mm Sample Preparation The mb sample was a gift from a local biotech company. Cytochrome C (equine), ribonuclease, lysozyme proteins, carboxypeptidae (CP), MES, NaCl and other chemicals were obtained from Sigma-ldrich. Part Number Samples were prepared and injected from Thermo Scientific ml Target DP polypropylene vials, 00 μl, C4000- with 9 mm VCS screw thread cap with pre-slitted silicone/ptfe septum C Carboxypeptidase Preparation: 5 mg sample was dissolved in ml deionized water and sub-aliquots were stored frozen (-0 C) until use. Mobile Phase for Protein nalysis Eluent : 0 mm MES (ph.4) Preparation: 0 mm MES (ph.4) was prepared by adding.9 g of MES to 900 ml of Milli-Q water. djust the ph with NaOH to.4 and volume to liter and filter before use. Eluent : 0 mm MES (ph.4) + M NaCl Preparation: 0 mm MES (ph.4) was prepared by adding.9 g of MES to 900 ml of Milli-Q water. djust the ph with NaOH to.4 and add g of NaCl, adjust volume to liter and filter before use. Mobile Phase for mb nalysis Eluent : Preparation: Eluent : Preparation: Eluent C: Preparation: 0 mm MES (ph 5.) + 0 mm NaCl 0 mm MES (ph 5.) is prepared by adding.9 g of MES to 900 ml of Milli-Q water. djust the ph with NaOH to 5. and add.50 g of NaCl, adjust volume to liter and filter (0. μm) before use. 0 mm MES (ph 5.) + 00 mm NaCl 0 mm MES (ph 5.) is prepared by adding.9 g of MES to 900 ml of Milli-Q water. djust the ph with NaOH to 5. and add 7.5 g of NaCl, adjust volume to liter and filter before use. 0 mm MES (ph 5.) + M NaCl 0 mm MES (ph 5.) is prepared by adding.9 g of MES (Sigma-ldrich) to 900 ml of Milli-Q water. djust the ph with NaOH to 5. and add g of NaCl, adjust volume to liter and filter (0. μm) before use. Data Processing Software: Thermo Scientific Dionex Chromeleon.8 Chromatography Data System

3 Results and Discussion ProPac WCX-0 and MbPac SCX-0 Phases for mb nalysis There are differences between the cation exchange phases of the ProPac WCX-0 and MbPac SCX-0 columns (Table ). The ProPac WCX-0 phase is a weak acid cation exchanger with a carboxylic function group, and the MbPac SCX-0 phase is a strong acid cation exchanger with a sulfonic acid functionality. They exhibit selectivity differences for these protein separations. oth phases are routinely used for high-resolution mb separations. Physical Characteristic ProPac WCX-0 MbPac SCX-0 Substrate Nonporous, highly cross-linked vinyl aromatic media Nonporous, highly cross-linked vinyl aromatic media Coating Hydrophilic layer Different hydrophilic layer Particle size 0 μm 0 μm, 5 μm, μm Grafting Conventional radical polymerization TRP Functional group Carboxylic acid Sulfonic acid Table : Physical characteristics of ProPac WCX-0 and MbPac SCX-0 phases When a three protein mixture was separated on these columns, under similar conditions, ribonuclease eluted first on the MbPac SCX-0 column, followed by cytochrome C and lysozyme. Conversely, cytochrome C eluted first on the ProPac WCX-0 column, followed by lysozyme and ribonuclease (Figure ), demonstrating selectivity differences between the two column chemistries. U Column:. MbPac SCX-0. ProPac WCX-0 Dimension: 4 50 mm Eluent : 0 mm MES, ph =.4 Eluent : 0 mm MES,.0 M NaCl, ph =.4 Gradient: Time(min) % % Flow Rate:.0 ml/min Inj. Volume: 0 μl Detection: UV at 54 nm U. Ribonuclease mg/ml. Cytochrome C 0. mg/ml. Lysozyme 0.5 mg/ml Figure : Selectivity differences of MbPac SCX-0 and ProPac WCX-0 columns Monoclonal ntibody nalysis oth the ProPac WCX-0 and MbPac SCX-0 columns are routinely used for separation of monoclonal antibodies and their variants. oth salt gradients and ph gradients can be used for the separation of mbs. detailed method is described and applications using ph gradients has been previously described [7].

4 4 High Throughput and High Resolution by Small Particle Columns comparison of mb separations using the ProPac WCX-0 column and the alternative selectivity MbPac SCX-0 column in a variety of particle sizes is shown in Figure C 7 D 7.5 cidic variants asic variants Columns: : ProPac WCX-0, 0 μm, 4 50 mm : MbPac SCX-0,0 μm, 4 50 mm C: MbPac SCX-0, 5 μm, 4 50 mm D: MbPac SCX-0, μm, 4 50 mm Eluent : 0 mm MES + 0 mm NaCl ph 5. Eluent : 0 mm MES + 00 mm NaCl, ph 5. Gradients: : % in 50min : 5.44% in 50min C: 0 5% in 0min D: 7% in 0min Flow Rate: ml/min (0.7mL/min for D) Temp.: 0 C Detection: Peaks: mb; 50 μg (0 μl) for & and 5 μg (5 μl) for C&D 80 nm Lysine variants,,, ; acidic variants and basic variants are shown Figure : Comparison of mb analysis with ProPac WCX-0 and MbPac SCX-0 cation exchange columns The 0 μm, 4 50 mm columns require a gradient of 50 minutes for well-resolved separation of variants. y comparison, and 5 μm, 4 50 mm columns require much shorter gradients of 0 minutes to produce comparable chromatography. These smaller particle size columns compensate for the longer length required by the 0 µm particle column m 0 m Column: 4 50 mm Eluent : 0 mm MES + 0 mm NaCI, ph 5. Eluent : 0 mm MES + 00 mm NaCl, ph 5. Gradient: 7% in 5 min ( & 5 μm) 0 5% in 5 min (0 μm) Flow Rate: 0.7 ml/ min Inj. Volume: 5 μl mb, 5 mg/ml Resolution values are shown in the inset for lysine truncation variants Particle size Peak Peak Peak μm μm μm m Figure : Comparison of mb separation on different particle size MbPac SCX-0 columns Figure shows the improved resolution of an mb separation on a small particle column when compared to a larger particle column. Column dimensions are kept constant in this example. Using the same flow rate (0.7 ml/min) and gradient slope, resolution values for three lysine truncation variants of an mb are assessed on 0 μm, 5 μm and μm particle size columns. Results show that when compared to a 0 μm column for lysine truncation, the resolution of peaks,, and is improved by %,.7%, and 7%, respectively, for 5 μm columns and 4%, 0.7% and 7%, respectively, for μm columns. Even at a flow rate of ml/ min, a 5 μm particle 4 x 50 mm column produces higher resolution of %, %, and 80% for lysine truncation peaks, and, respectively, when compared to a 0 μm particle column (Figure 4).

5 cidic variants asic variants Columns: : MbPac SCX-0, 5 μm, 4 50 mm : MbPac SCX-0, 0 μm, 4 50 mm Eluents: ) 0 mm MES + 0 mm NaCI, ph 5. ) 0 mm MES + 00 mm NaCl, ph 5. C) 0 mm MES + M NaCl, ph 5. Gradients: 0 5% in 5 min Eluent: C wash: min Eluent C wash before equilibration Equilb. Time: 4 min Flow Rate: ml/min Inj. Volume: 5 μl Temp.: 0 C mb, 5 mg/ml Detection: UV at 80 nm Peaks:,, : C-terminal lysine truncation variants. Resolution is shown in inset. 5 Particle size Peak Peak Peak 5 μm μm Figure 4: Comparison of mb analysis on MbPac SCX-0 0 μm, and 5 μm columns at high flow rate of ml/min High-Throughput nalysis Using a Small Particle Column; Monitoring Processing of C-terminal Lysine Residues of mbs Charge heterogeneity can result if the processing of C-terminus of a protein is incomplete [5]. The resulting charge heterogeneity of the variant forms can be identified by cation exchange chromatography. In this example, the MbPac SCX-0 column was used to separate variants of a mb containing lysine residue variation at the C-terminal of the heavy chains. comparative analysis was performed on a 0 μm MbPac SCX-0, 4 50 mm column (Figure 5) and a μm MbPac SCX-0, 4 50 mm column (Figure 5) using different flow rates and gradient conditions. For the 0 μm, 50 mm column, a 50 minute gradient time was used, which is considerably shortened to a 5 minute gradient time in the case of the μm column. y doing this, overall run time was decreased by more than five-fold and resulted in increased throughput with the small particle column. In both cases, three variant forms differing by the presence of lysine at the C-terminal of the heavy chains with either no lysine, or lysine, or lysine residues are separated as individual peaks (Figure 5 and ; sample ). To verify that the different retention times of the three peaks were due to the different heavy chain C terminal lysine content, the mb was treated with carboxypeptidase. This is an exopeptidase that specifically cleaves C terminal lysine residues (Figure 5 and ; sample ). This treatment of the mb sample resulted in the absence of peaks 7 and 8 (containing and terminal lysine residues, respectively, on their heavy chains). The decreased peak areas in peaks 7 and 8 are accompanied by a corresponding increase in area of peak where no lysine is present. nother minor variant with lysine truncations, peaks 9-, collapsed to peak 9 after carboxypeptidase treatment (Figure 5 and ). High-throughput monitoring of C-terminus lysine truncations on a small particle μm column is shown in Figure 5. Total analysis time is reduced more than five-fold for the μm column when compared to the 0 μm, 4 50 mm column.

6 Eluents: : 0 mm MES (ph 5.) + 0 mm NaCl : 0 mm MES (ph 5.) + 00 mm NaCl Temp: 0 C Detection:. Column: Gradient: Flow Rate: Inj. Volume: 80 nm. mb. mb + Carboxypeptidase (CP) MbPac SCX-0, 0 μm, 4 50 mm. mb, 9 mg/ml. 0.9 mg of mb was treated with 50 μg of CP. Final mb concentration was 8. mg/ml 5.44% in 50 min ml/min 5 μl. Column: Gradient: Flow Rate: Inj. Volume: Peaks MbPac SCX-0, μm, 4 50 mm. mb,.5 mg/ml. 0.5 mg of mb was treated with 50 μg of CP. Final mb concentration was. mg/ml. 7 % in 5 min 0.5 ml/min μl 5: cidic variants; Peaks, 7, 8: C-terminal Lys truncation variants of main peak; Peaks 9, 0, : C-terminal Lys truncation variants of a minor variant peak Figure 5: Characterization of mb C-terminal lysine truncation variants using MbPac SCX-0 0 μm (Panel ) or μm (Panel ) columns. μm, 4 50 mm column separation is more than five times faster than MbPac SCX-0 0 μm, 4 50 mm column. (note that time scales are different) Ruggedness of MbPac SCX-0 Small Particle Columns Figure demonstrates that more than 0 runs were performed without significant loss of resolution. Peak widths at half height values were determined for lysine truncation variants (Table ). Fast, 5 minute gradients were used in this assessment. The ruggedness of the column improved if columns were washed with a high salt concentration buffer after each run. 7 Run # 4 Run # 49 5 Run # 9 4 Run # 09 Run # 74 Run # 44 Run # 9 Run # 99 Run # 79 Run # 57 Run # Figure : Ruggedness of MbPac SCX-0 μm, 4 50 columns Columns: MbPac SCX-0, μm, 4 50 mm Eluents: ) 0 mm MES + 0 mm NaCI, ph 5. ) 0 mm MES + 00 mm NaCl, ph 5. C) 0 mm MES + M NaCl, ph 5. (for wash) Gradients: 5% in 5 min Eluent C Wash: min Eluent C wash before equilibration Equilib. Time: 4 min Flow Rate: 0.8 ml/min Inj. Volume: 5 μl Temp.: 0 C Detection: UV at 80 nm mb, 5 mg/ml (Injected intermittently at every to 5 runs; Peaks:,, : C-terminal lysine truncation variants. PWHH values are shown in Table

7 Peak Peak Peak verage RSD (%).0.. Table : Ruggedness of MbPac SCX-0 μm columns pplication Note 008 Conclusion This application note demonstrates high-throughput, high-resolution mb separation applications using MbPac SCX-0 small particle columns. References [] Weitzhandler, M.; Farnan, D.; Horvath, J.; Rohrer, J.S.; Slingsby, R.W,; vdalovic, N.; Pohl, C. Protein variant separations by cation-exchange chromatography on tentacle-type polymeric stationary phases. Journal of Chromatography, 998, 88, 5-7. [] Moorhouse, K.G.; Nashabeh, W.; Deveney, J.; jork, N.S.; Mulkerrin, M.G.; Ryskamp, T. Validation of an HPLC method for the analysis of the charge heterogeneity of the recombinant monoclonal antibody IDEC-C8 after papain digestion. Journal of Pharmaceutical and iomedical nalysis, 997,, [] Harris, R.J.; Kabakoff,.; Macchi, F.D.; Shen, F.J.; Wong, M.K.; ndya, J.D.; Shire. S.J.; jork, N.; Totpal, K.; Chen,.. Identification of multiple sources of charge heterogeneity in a recombinant antibody. Journal of Chromatography, 00, 75, -45. [4] Farnan, D. and Moreno, G.T. Multiproduct high-resolution monoclonal antibody charge variant separations by ph gradient ion-exchange chromatography. nalytical Chemistry, 009, 8, [5] Harris, R.J. Processing of C-terminal lysine and arginine residues of proteins isolated from mammalian cell culture. Journal of Chromatography, 995, 705, 9-4. [] Vlasak, J. and Ionescu, R. Heterogeneity of monoclonal antibodies revealed by charge-sensitive methods. Current Pharmaceutical iotechnology, 008, 9, [8] Lin, S.; aek, J.; Rao, S.; groskin, Y.; Pohl, C.; Decrop, W.; Milton, D. novel ph gradient separation platform for monoclonal antibody (Mb) charge variant analysis. Thermo Scientific pplication Note 0784, 0. thermoscientific.com/columns 0 Thermo Fisher Scientific Inc. ll rights reserved. Milli-Q is a registered trademark of MERCK KGa. Sigma-ldrich is a registered trademark of Sigma-ldrich Co, LLC. ll other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is presented as an example of thecapabilities of Thermo Fisher Scientific products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. US and Canada ustralia (free call domestic) China (free call domestic) France + (0) Germany +49 (0) or India Japan (free call domestic) fax United Kingdom +44 (0) New Zealand (free call domestic) Singapore ll Other Enquiries +44 (0) Technical Support For advice and support, please visit our website: N008-EN 0S