School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland.
J Sep Sci. 2012 Nov;35(22):3113-23. doi: 10.1002/jssc.201200297. Epub 2012 Jun 20.
Recent reversed-phase wide-pore stationary phases were evaluated for the separation of intact monoclonal antibodies and their fragments. Two types of stationary phases were tested: Phenomenex Aeris Widepore, with 3.6 μm core-shell particles and Waters Acquity BEH300 with 1.7 μm fully porous particles. A systematic investigation was carried out using model IgG1 and IgG2 antibodies, namely rituximab, panitumumab, and bevacizumab. It appeared that adsorption of these antibodies on the stationary phase was significantly higher compared to proteins of equivalent size. The adsorption was particularly important for the intact antibodies of 150 kDa and for the largest fragments of 50 to 100 kDa (i.e., heavy chain, -fraction of antigene-binding). The present study demonstrated an obvious relationship between adsorption phenomenon and the unwanted strong secondary interactions (ionic and hydrogen bond) of the stationary phase. Thus, adsorption was more pronounced on the Aeris column because of the stronger ion exchange contribution of this stationary phase. Using C4 phase instead of C18 at 50-70°C, there is a slight reduction (5-20%) in adsorption. Two solutions were proposed to decrease the strength of secondary interactions and thus resolve (or at least diminish) adsorption issue. First, increasing mobile phase temperature up to 80-90°C appeared as a promising solution. However, temperature should be used with caution as it can partially damage large biomolecules. A compromise between residence time and temperature should be found. Second, it is recommended to add a small amount of an ancillary solvent, such as n-butanol to the mobile phase. Indeed, the hydroxyl group of n-butanol probably interacts with water adsorbed on the residual silanol groups "to shield" silanols.
最近，研究人员评价了几种反相宽孔径固定相，以分离完整的单克隆抗体及其片段。研究人员测试了两种类型的固定相： Phenomenex Aeris Widepore，采用 3.6μm 核壳颗粒；Waters Acquity BEH300，采用 1.7μm 全多孔颗粒。使用模型 IgG1 和 IgG2 抗体（即利妥昔单抗、帕尼单抗和贝伐珠单抗）进行了系统研究。结果表明，与同等大小的蛋白质相比，这些抗体在固定相上的吸附量明显更高。对于 150kDa 的完整抗体和 50-100kDa 的最大片段（即重链、抗原结合的-fraction），这种吸附尤其重要。本研究表明，吸附现象与固定相的不必要的强次级相互作用（离子和氢键）之间存在明显的关系。因此，由于 Aeris 柱的离子交换贡献更强，吸附现象更为明显。在 50-70°C 下，使用 C4 相代替 C18 相，吸附减少了 5-20%。提出了两种解决方案来降低次级相互作用的强度，从而解决（或至少减少）吸附问题。首先，将流动相温度提高到 80-90°C 似乎是一种有前途的解决方案。然而，应谨慎使用温度，因为它可能会部分损坏大生物分子。应在保留时间和温度之间找到一个折衷方案。其次，建议在流动相中添加少量辅助溶剂，如正丁醇。事实上，正丁醇的羟基可能与吸附在残留硅醇基团上的水相互作用，“屏蔽”硅醇基团。
