Independent Study. Column Selection IEX

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1 YMC-BioPro SP-F Page 1/6

2 Overview The Universities of Geneva and Lausanne performed a comparative examination of dissimilar IEX columns of different manufacturers within an independent study. The focus was on the following chromatographic parameters: Selectivity Resolution YMC also participated with YMC-BioPro SP-F, which is a strong non-porous cation exchanger. Conclusion The capacity of CEX columns for therapeutic monoclonal antibodies (mabs) was chromatographic analysed in the ph- and salt-gradient mode. YMC-BioPro SP-F scoring the highest selectivity and resolution (see table 1)! Table 1: Cation exchange columns Column Manufacturer CEX Particle size [µm] Column length [mm] YMC-BioPro SP-F YMC Strong (sulfonic) MAbPac SCX-10 Thermo Scientific Strong (sulfonic) Protein-Pak Hi Res SP Waters Strong (sulfonic) Antibodix WCX-NP3 Sepax Weak (carboxylate) Bio Mab NP Agilent Weak (carboxylate) These four mabs were selected as examples for their chromatographic properties: 1. Natalizumab (peak 1) -> few interactions -> short retention time YMC-BioPro SP-F convinces with the narrowest peak width. 2. Cetuximab as a complex heterogeneous mab (peak 2) -> secondary interactions impede the isolation. YMC-BioPro SP-F enables the resolution even for demanding isolations. 3. Adalimumab (peak 3) and denosumab (peak 4) possess some unique retention behaviour. YMC-BioPro SP-F achieves the greatest selectivity using the salt gradient mode. Page 2/6

3 Study results Separation power Figure 1 shows the chromatograms obtained on YMC-BioPro SP-F, Antibodix WCX NP3, MabPac SCX-10RS and on Protein-Pak Hi Res SP columns in ph and salt gradient modes for natalizumab, cetuximab, adalizumab and denosumab. In figure 1 the results implying the ph gradient mode are shown. Figure 2 presents the chromatograms with a salt gradient mode. YMC-BioPro SP-F Narrowest peak width! Best separation! Protein-Pak Hi Res SP Antibodix WCX-NP3 MabPac SCX-10 RS Figure 1: Representative chromatograms of natalizumab (1), cetuximab (2), adalimumab (3) and denosumab (4) obtained on different columns in the ph gradient mode. Page 3/6

4 YMC-BioPro SP-F Greatest selectivity! Protein-Pak Hi Res SP Antibodix WCX-NP3 MabPac SCX-10 RS Figure 2: Representative chromatograms of natalizumab (1), cetuximab (2), adalimumab (3) and denosumab (4) obtained on different columns in the salt gradient mode. Page 4/6

5 High resolution CEX by Efficient columns are required when dealing with therapeutic mab samples to obtain high peak capacities [2]. A approach could help to improve antibody CEX analysis. This allows to compare the chromatographic profiles of different batches or bio-similar products. Because YMC-BioPro SP-F showed the highest resolution for the separation of natalizumab, cetuximab, adalimumab and denosumab, it was selected to perform high resolution separation by using a approach (see Figure 3). Figure 3 illustrates the improved resolution of the different variants through the extension of column length and gradient time. Cetuximab Adalimumab Column length Resolution increases Gradient time Figure 3: The impact of column length and gradient time (gradient steepness) on the resolving power in the salt gradient mode using YMC-BioPro SP-F ( mm, 5 µm). Page 5/6

6 As shown in this study, the YMC-BioPro SP-F column is an outstanding stationary phase for the characterisation of charge variants mabs in CEX mode. References [1] S. Fekete, A. Beck, D. Guillarme, Characterisation of cation exchanger stationary phases applied for the separations of therapeutic monoclonal antibodies, J. Pharm. Biomed. Anal., 2015, 111, [2] S. Fekete, M. Dong, T. Zhang, D. Guillarme, High resolution reversed phase analysis of recombinant monoclonal antibodies by ultra-high pressure liquid chromatography, J. Pharm. Biomed. Anal., 2013, 83, Page 6/6