MAbPac SCX-10 Column for Monoclonal Antibody Variant Analysis and Characterization

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1 CHROMTOGRPHY MbPac SCX- Column for Monoclonal ntibody Variant nalysis and Characterization Product Specifications The Thermo Scientific MbPac SCX- columns separate closely-related monoclonal antibody variants for characterization and quality control assessment. High resolution of monoclonal antibody variants Exceptionally high efficiency vailable in, or μm particle size PEEK and UHPLC column formats High throughput capability Excellent lot-to-lot and column-to-column reproducibility Ideal for R&D and Q/QC development High-Resolution, High-Efficiency nalysis of Monoclonal ntibody (mb) Variants The MbPac SCX- column is a strong cation-exchange column designed specifically for the high resolution, high efficiency analysis of monoclonal antibodies and associated variants. The unique nonporous pellicular resin provides high resolving power, permitting the separation of monoclonal antibody variants that differ by as little as one charged residue. The MbPac SCX resin technology is the basis for the superior performance of monoclonal antibody variant analysis. The nonporous core particle provides high rates of mass transfer, which results in high efficiency separations. proprietary hydrophilic layer surrounds the polymeric beads preventing hydrophobic interactions between proteins and the core of the resin. proprietary grafted cation-exchange surface provides an ideal separation platform for high-resolution separations. MbPac SCX- small particle columns are available in PEEK and PEEKlined stainless steel (RS series of columns) housings. While PEEK columns are available in three different particle sizes of µm, µm and µm, PEEK-lined stainless steel columns are currently available in µm particle sizes. Depending on the resolution desired and the time allotted for specific applications,, or µm columns may be used. For HPLC based applications, PEEK columns are suitable and are recommended. For UHPLC applications, PEEK-lined stainless steel RS columns are recommended as they are compatible with high pressure and can withstand pressures up to, psi. Higher flow rates may be used with Rapid Separation (RS) columns when compared to PEEK columns, for faster run time and to achieve high throughput separations. RS version of the MbPac SCX- columns are built with PEEK-lined stainless steel. These columns are designed to be used with bioinert UHPLC system.

2 MbPac SCX-, µm and MbPac SCX-, µm columns are recommended for high resolution and high-throughput separations of mbs. The main advantage being the run time is reduced when or µm, shorter columns are used as compared to a longer MbPac SCX-, µm, 4 mm columns. Since the column length is short, chromatography runs can be completed at a faster rate, therefore increasing the throughput. The MbPac SCX- column is complementary to the industry-leading Thermo Scientific ProPac WCX- column for monoclonal antibody variant analysis, offering an alternative selectivity and providing higher resolution and efficiency for variant analysis of monoclonal antibody samples. Selectivity Differences etween ProPac WCX- and MbPac SCX- MbPac SCX columns exhibits different selectivity as compared with ProPac WCX for protein separations. When a protein mixture was separated on these columns under identical conditions, ribonuclease elutes first on the MbPac SCX column, followed by cytochrome C and lysozyme. In comparison, cytochrome C elutes first on ProPac WCX column followed by lysozyme and ribonuclease (Figure and ), confirming the selectivity changes. Characterization of Monoclonal ntibody Heterogeneity; cidic and asic Variant nalysis Monoclonal antibodies are currently developed by pharmaceutical and biotechnology companies for various therapeutic applications. They undergo several modifications including oxidations, deamidations, glycosylation, incomplete C-terminal processing, and others ( ). These modifications cause antibody microheterogeneity. Variations in a monoclonal antibody s composition can impact its activity and stability as a biotherapeutic. Monitoring stability of therapeutic monoclonal antibodies is regarded as essential for demonstrating safety and efficacy of a monoclonal antibody drug, and is expected by the FD and other regulatory agencies. The MbPac SCX- column is useful for characterizing monoclonal heterogeneity, and for stability testing.. U U C - 4 Columns:. MbPac SCX-, μm, 4 mm. ProPac WCX-, μm, 4 mm Eluents:. mm MES, ph =.4. mm MES,. M NaCl, ph =.4 Gradient: Time(min) % % D Flow Rate: ml/min Inj. Volume: µl Detection: UV at 4 nm Sample:. Ribonuclease mg/ml. Cytochrome C. mg/ml. Lysozyme. mg/ml 4 Figure : The MbPac SCX- exhibits selectivity differences from ProPac WCX- (Figure and ) when separating proteins. dditionally, MbPac SCX- provides excellent peak separation and resolution for different mbs (Figure C and D) Column: MbPac SCX-, μm, 4 mm Eluents:. mm MES (ph.) + mm NaCl. mm MES (ph.) + mm NaCl Gradient: % in min, gradient slope constant Flow Rate: ml/min Inj. Volume: µl Detection: UV at nm Sample: mb, mg/ml Column: MbPac SCX-, μm, 4 mm Eluents:. mm MES (ph.) + mm NaCl. mm MES (ph.) + mm NaCl Gradient: % in min Flow Rate: ml/min Inj. Volume: µl Temp.: C Detection: UV at nm Sample: mb, mg/ml Peaks : cidic variants Peaks,, : C-Terminal Lys variants Peaks : asic variants

3 One of the most important and common analyses of monoclonal antibody heterogeneity is the monitoring and determination of acidic and basic variants. The MbPac SCX- column provides high resolution for variant analysis of monoclonal antibodies, where two different monoclonal antibodies are separated (Figure C, D).. Column: MbPac SCX-, μm, 4 mm Eluents:. mm MES (ph.) + mm NaCl. mm MES (ph.) + mm NaCl Gradient: % in min Flow Rate: ml/min Inj. Volume: µl Temp.: C Detection: UV at nm Samples:. mb, µg in µl (no CP). mb, µg in µl + CP, µg, incubation at C for h oth Chromatograms: Peaks : cidic variants Sample : Peaks : C-Terminal lysine truncation variants of main peak Peaks : C-Terminal lysine truncation variants of minor variant peak Sample : Peak results from peaks,, and after CP treatment Peak results from peaks,, and after CP treatment C-Terminal Lysine Variant nalysis During the development and the production of therapeutic monoclonal antibodies, characterization of structural variants is a critical challenge. C-terminal processing of lysine residues on the heavy chain of monoclonal antibodies is a common structural variation that demands analysis. Incomplete monoclonal antibody processing results in charge heterogeneity, which is readily identified using the MbPac SCX- column. Figure illustrates this point with excellent resolution of C-terminal lysine truncation variants, and other acidic and basic variants of a monoclonal antibody. 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, sample ). This treatment of the mb sample resulted in the absence of peaks and (containing and terminal lysine residues, respectively, on their heavy chains). The decreased peak areas in peaks and are accompanied by a corresponding increase in area of peak where no lysine is present. nother minor variant with lysine truncations, Peaks ) collapsed to peak after carboxypeptidase treatment. nalysis of mb Fragments fter Digestion with Papain and Carboxypeptidase Monoclonal antibodies treated with papain enzyme are separated into their Fab and Fc fragments. Figure shows the well resolved Fab and Fc fragments after a monoclonal antibody and its variants are treated with papain alone, or with papain and carboxypeptidase together. The expected acidic, C-terminal lysine truncationcontaining Fc fragments and Fab fragment peaks are determined and well resolved. Lysine truncation variant peaks collapse into one main peak with the carboxypeptidase treatment Figure : aseline resolution of C-terminal lysine variants of a monoclonal antibody sample. second chromatogram verifies that the three major peaks are due to variations in C-terminal content: after the treatment with CP, only one major peak remains. Column: Eluents: Gradient: Flow Rate: Inj. Volume: µl Temp.: C MbPac SCX-, μm, 4 mm. mm MES (ph.) + mm NaCl. mm MES (ph.) + mm NaCl % in min ml/min 4 Figure : High resolution analysis of monoclonal antibody fragments after treatment with papain or papain and CP enzymes. The expected acidic, C-terminal lysine variants containing Fc, and Fab fragment peaks are well resolved. 4 Detection: UV at nm Samples:. mb, mg in µl + papain, µg. mb, mg in µl + papain, µg + CP, µg, incubation at C for h oth Chromatograms: Peaks 4: cidic variants Sample : Peaks : C-Terminal lysine truncation variants of papain-treated sample Peaks, 4: Fab peaks Sample : Peak results from Sample peaks,, and after CP treatment Peaks, : Fab peaks

4 4 Optimizing the Separation with ph Gradients ph gradient can also be used for monoclonal antibody variant separations. The isoelectric point (pi) is the ph at which a particular protein carries no net charge and can no longer bind to the charged surface and therefore elutes. ph gradients are becoming increasingly popular to ease the method development process (4-). The recently introduced Thermo Scientific CX- ph Gradient uffers meet the fast and robust platform method requirement.this buffer system consists of a low-ph buffer at ph. and a high-ph buffer at ph.. linear ph gradient from ph. to ph. is generated over time by running a linear pump gradient from % buffer to % buffer (Figure 4). This is followed by a shallow gradient run, to optimize the mb separation (Figure 4). Fast mb Characterization nalysis MbPac SCX-, μm, mm columns provide high resolution monoclonal antibody separation when compared to longer MbPac SCX-, or ProPac WCX-, μm columns but with significantly faster analysis time (Figure ). Total analysis time is reduced nearly by fold for the μm column (Figure C) when compared to μm, 4 mm columns Figure and ). Column: MbPac SCX-, μm 4 mm Eluents: X CX-, ph Gradient uffer (ph.) X CX-, ph Gradient uffer (ph.) Gradients: % in minutes % in minutes Flow Rate:. ml/min Inj. Volume: µl Sample: mb, mg/ml ph trace ph trace Figure 4: Separaton of monoclonal antibody variants using ph gradient on a MbPac SCX-, μm, 4 mm column Columns: ) ProPac WCX-, μm 4 mm ) MbPac SCX-, μm 4 mm C) MbPac SCX, µm, 4 mm Eluents: : mm MES + mm NaCl, ph. : mm MES + mm NaCl, ph. Gradients: ) 4.44% in min ).44% in min C) % in min Flow Rate: ml/min (. ml/min for C) 4 Inj. Volume: and : µl ( µg) C: µl ( µg) Temp.: C Detection: nm Sample: mb; and ) mg/ml C) mg/ml Peaks: : cidic variants;,,: Lysine truncation variants : asic variants ph ph C C 4 4 Figure : The μm MbPac SCX-, 4 mm column provides significantly faster analysis while maintaining the similar high resolution as that obtained using the MbPac SCX- and ProPac WCX-, 4 mm columns

5 Figure demonstrates MbPac SCX-, μm column providing fast, monoclonal antibody variant analysis using MES-based salt gradients. (Figure ). Higher resolution is achieved with a longer minute gradient (Figure ) when compared to a minute gradient. comparative lysine truncation verification analysis was performed on a μm MbPac SCX-, 4 mm column (Figure ) and a μm MbPac SCX, 4 mm column (Figure ) using different flow rates and gradient conditions. 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 is separated as individual peaks (Figure and ; sample ). Please note that time scales are substantially different. To verify that the reason for the different retention times of the three peaks was the different content of heavy chain C terminal lysine, the mb was treated with carboxypeptidase, an exopeptidase that specifically cleaves C terminal lysine residues (Figure and, sample ). This treatment of the mb sample resulted in the quantitative disappearance of peaks and (containing and terminal lysine residues, respectively, on their heavy chains). The decreased peak areas in peaks and are accompanied by a corresponding increase in peak area of peak where no lysine is present (Peak area values not shown). Similarly, another minor variant with lysine truncations shown as Peaks, and collapsed to peak after carboxypeptidase treatment. High throughput advantage of a small particle μm column for monitoring C-terminus lysine truncations is shown in. Total analysis time is reduced nearly by fold for μm column when compared to μm, 4 mm column. Columns: MbPac SCX-, µm, 4 mm Eluents: : mm MES + mm NaCI, ph. : mm MES + mm NaCl, ph. Gradients: ) % in min ) % in min Flow Rate: ml/min Inj. Volume: µl Figure : The μm MbPac SCX-, 4 mm column provides fast, high resolution monoclonal antibody variant analysis Eluents: : mm MES (ph.) + mm NaCl : mm MES (ph.) + mm NaCl Temp: C Detection: nm Sample:. mb. mb + Carboxypep.dase (CP). Column: MbPac SCX-, μm, 4 mm Mb:. mg/ μl +/- μg CP in μl Gradient:.44% in min Flow Rate: ml/min Inj. Volume: μl K Temp.: C Sample: mb, mg/ml Detection: UV at nm Peaks:. Column: Mb: Gradient: Flow Rate: Inj. Volume: K K 4. cidic variants,,. C-Terminal lysine variants -. Other basic variants MbPac SCX-, μm, 4 mm. mg/ μl+/- μg CP in ul % in min. ml/min μl Peaks : cidic variants; Peaks,, : C- terminal Lys trunca.on variants of main peak; Peaks,, : C- terminal Lys trunca.on variants of a minor variant peak K K K Figure : Characterization of mb C-terminal lysine truncation variants using MbPac SCX-, µm (Panel ) or µm (Panel ) columns. µm, 4 mm column separation is more than times faster than MbPac SCX-, µm, 4 mm column. Note: timescales are different.

6 UHPLC mb Separation High resolution analysis of mb charge variants is achieved using UHPLC based separations. ioinert PEEK-lined stainless steel column housings are used for these Rapid Separation (RS), small particle columns. These columns take advantage of small resin size as well as longer column length to maximize the resolution of mb variant separation. They are suitable for operation up to the higher pressure limit of, psi. MbPac SCX- RS, μm columns are designed to be used with bioinert UHPLC system; Thermo Scientific UltiMate iors totally inert UHPLC system is recommended for best results. The RS columns are available in 4. mm I.D. as well as. mm I.D to suit different applications. There are several advantages of using. mm I.D. columns including sample and eluent conservation. However, it should be noted that the same gradient method developed with the 4. mm column might not work with a. mm format column. This is especially evident when using lower flow rates due to influence of instrument delay volume. It is important to re-establish the optimal gradient run conditions for. mm formats. oth salt gradients and/or ph gradients can be used for high resolution separation of mbs. Salt Gradients Figure shows an example of mb separation comparing MbPac SCX- RS, μm, 4. mm with 4. mm columns at. ml/min flow rate ( and C). oth columns resolved acidic and basic variants from the main lysine truncation peaks. Resolution values of lysine truncation peaks are shown. longer 4. mm column produces high resolution when compared to a 4. mm column ( Figure and C). Comparison of different flow rates was made using MbPac SCX- RS, μm, 4. mm column (Figure ). High pressure compatibility of the column allows the use of high flow rates ( ml/min) and short run times while maintaining decent resolution. This capability enables high throughput characterization of samples. Figure shows the separation of a mb using a salt gradient on MbPac SCX- RS, μm,. mm mm column. s expected a shallow gradient (Figure ) produces higher resolution when compared to a steep gradient (Figure ). & Column: MbPac SCX- RS, μm Dimension: 4. mm Sample: mb mg/ml Inj. Volume: μl Eluent : mm MES ph (.) Eluent : mm MES ph (.) + mm NaCl Flow Rate: Gradient: C. ml/min % in min cidic variants Figure : Mb separation on MbPac SCX- RS, μm columns, comparison of different flow rates and different lengths of RS columns Column: MbPac SCX- RS, μm Dimension:. mm Eluent : mm MES ph (.) Eluent : mm MES ph (.) + mm NaCl Gradients: % in.min (Panel ) % in min (Panel ) Flow Rate:. ml/min Inj. Volume:. μl Sample: mb mg/ml Resolution values are shown for Lysine truncation variants Figure : Mb separation on MbPac SCX- RS, μm. mm column using different gradient conditions... n.a. Lysine truncation variants.4 Other basic variants Flow Rate: Gradient: C Column: Dimension: Gradient: Flow Rate: Inj. Volume: Sample: ml/min % in min MbPac SCX- RS, μm 4. mm % in. min. ml/min μl mb mg/ml Resolution values are shown for Lysine truncation variants.4 n.a.

7 Faster ph Gradients Faster ph gradient separations can be achieved using the RS format: MbPac SCX- RS column. Figure shows a separation using a μm, 4. mm mm column completed in half the time of a μm column. Similar to the μm separation described in Figure 4, the ph gradient can be first run with a broad gradient (Figure ) and then optimized for greater peak resolution with a narrow gradient (Figure ). Column: MbPac SCX- RS, μm 4. mm Eluent : X CX-, ph Gradient uffer (ph.) Eluent : X CX-, ph Gradient uffer (ph.) Gradient: % in minutes (Panel ) % in minutes (Panel ) Flow Rate:. ml/min Inj. Volume: μl Sample: mb, mg/ml - ph trace ph Higher sensitivity, with lower eluent consumption, can be achieved using the. mm RS format MbPac SCX- RS column, as is shown in Figure. Panel shows the initial broad separation, whereas Panel shows a optimized narrow gradient.... ph trace ph Figure : Faster separation of Monoclonal ntibody Variants using ph gradient on a MbPac SCX- RS, μm, 4. mm column Column: MbPac SCX- RS, μm Dimension:. mm Eluent : X CX-, ph Gradient uffer (ph.) Eluent : X CX-, ph Gradient uffer (ph.) Gradient: % in minutes (Panel ) % in minutes (Panel ) Flow Rate:. ml/min Inj. Volume: μl Sample: mb, mg/ml Peak Resolution values are shown Figure : Higher sensitivity separation of Monoclonal ntibody Variants using ph gradient on a MbPac SCX- RS, μm,. mm column

8 Ruggedness of MbPac SCX- Columns Ruggedness of MbPac SCX- RS, μm, 4. mm column was demonstrated. This was evaluated at ml/min flow rate for more than runs Figure ). Peak width data obtained for the lysine truncation peaks is shown in Table. Column: MbPac SCX-, μm, 4. mm Eluent: : mm MES (ph.) : mm MES (ph.) + mm NaCl Gradient: % in min Flow Rate: ml/min 4 - Run Inj. Volume: μl Temp: C Detection: nm Sample: mb, mg/ml Sample was injected intermittently Run Run 4 Run Run Run 4 Run Run Run 4 Run Figure : Ruggedness testng of MbPac SCX- RS, μm, 4. mm column. t flow rate of ml/minutes lysine truncation peaks are identified as,, peaks and their Peak Width Half Height data (in minutes) is given overpage. Sample No Peak Peak Peak verage... RSD (%) Table : Ruggedness testing of MbPac SCX- RS, μm, 4. mm column at flow rate of ml/min (Figure ). Peak Width at Half Height values in minutes are shown for Lysine truncation peaks.

9 References [] K.G. Moorhouse, W. Nashabeh, J. Deveney, N. S. jork, M. G. Mulkerrin, T. Ryskamp, Validation of an HPLC method for the analysis of the charge heterogeneity of the recombinant monoclonal antibody IDEC-C after papain digestion, Journal of Pharmaceutical and iomedical nalysis () -. [] R. J. Harris,. Kabakoff, F. D. Macchi, F.J. Shen, M. K wong, J. D. ndya, S. J. Shire, N.jork, K. Totpal,.. Chen, Identification of multiple sources of charge heterogeneity in a recombinant antibody, Journal of Chromatography : iomedical Sciences and pplications () -4. [] R. J. Harris, Processing of C-terminal lysine and arginine residues of proteins isolated from mammalian cell culture, Journal of Chromatography () -4. [4] D. Farnan, G. T. Moreno, Multiproduct high-resolution monoclonal antibody charge variant separations by ph gradient ion-exchange chromatography, nalytical Chemistry () 4-. [] J.C. Rea, G.T. Moreno, Y. Lou,and D. Farnan. Validation of a ph gradient-based ion-exchange chromatography method for high-resolution monoclonal antibody charge variant separations. J Pharm iomed nal. ; 4():- [] Specifications and Operational Parameters Column construction ead size Substrate Pellicular layer Functional group Pressure limit PEEK and PEEK lined stainless steel iors columns μm, μm, μm Nonporous, highly crosslinked vinyl aromatic media Proprietary hydrophilic Sulfonic PEEK Columns μm: psi μm and μm: psi RS Columns μm: psi Temperature range mbient to C ph range Dynamic loading capacity Recommended buffers Detergent compatibility Up to μg, depending on the monoclonal antibody sample ( μm, 4 mm) MES, or other Good s buffers, tris, Thermo Fisher Scientific ph gradient buffers CX-(pH.) and CX- (ph.). Nonionic, anionic, or zwitterionic detergents Dynamic loading capacity µm, 4 mm: µg RS µm, 4. mm: 4 µg (For a typical mb) µm, 4 mm: µg RS µm, 4. mm: µg µm, 4 mm: µg RS µm, 4. mm: µg µm, 4 mm: µg RS µm,. mm: µg µm, 4 mm: µg RS µm,. mm: 4 µg RS µm,. mm: 4 µg

10 Ordering Information MbPac SCX- nalytical Column MbPac SCX-, μm, nalytical Column (4 mm) MbPac SCX-, μm, nalytical Column (4 mm) MbPac SCX-, µm, nalytical Column (4 mm) MbPac SCX-, μm, nalytical Column (4 mm) MbPac SCX-, μm, nalytical Column (4 mm) MbPac SCX-, μm, nalytical Column (4 mm) 4 MbPac SCX-, μm, nalytical Column SCX-HT (4 mm) MbPac SCX-, μm, nalytical Column SCX- ( mm) 4 Product Specifications UHPLC, MbPac SCX- RS, nalytical Column MbPac SCX- RS, µm, nalytical Column (4. mm) 4 MbPac SCX- RS, µm, nalytical Column (4. mm) MbPac SCX- RS, μm, nalytical Column (4. mm) MbPac SCX- RS, µm nalytical Column (. mm) MbPac SCX- RS, µm nalytical Column (. mm) 4 MbPac SCX- RS, µm nalytical Column (. mm) Lot Select Column Set Lot Select Column Set Three columns from one resin lot (4 mm) Lot Select Column Set One column from each of three resin lots (4 mm) MbPac SCX- Semipreparative Column MbPac SCX-, μm, Semipreparative Column ( mm) 4 MbPac SCX- Guard Column MbPac SCX- G, μm, Guard Column ( mm) 4 MbPac SCX- G, μm, Guard Column (4 mm) 4 ph uffer Concentrates CX- ph Gradient uffer (ph.), ml CX- ph Gradient uffer (ph.), ml CX- ph Gradient uffer Kit (ph. to.), ml 4 CX- ph Gradient uffer (ph.), ml 4 CX- ph Gradient uffer (ph.), ml 4 CX- ph Gradient uffer Kit (ph. to.), ml 4 To find a local representative, visit: Thermo Fisher Scientific Inc. ll rights reserved. ISO is a trademark of the International Standards Organization. ll other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. PS-EN S