Analytical R&D, Global Biologics, Pfizer Inc., Chesterfield, MO 63017, USA.
J Sep Sci. 2010 Sep;33(17-18):2671-80. doi: 10.1002/jssc.201000230.
A rapid reversed-phase HPLC separation of recombinant human immunoglobulin gamma 2 (IgG2) disulfide isomers using columns packed with superficially porous particles is reported. Under optimal conditions, a separation of monoclonal IgG2 disulfide isomers was achieved in 10 min using a Poroshell™ 300SB-C8 column via a combination of high column temperature (85°C), mobile phases with high eluotropic strength (e.g. isopropanol) and high flow rate (1.5 mL/min). Thermodynamic stability analyses of chromatographically enriched IgG2 disulfide isomers revealed differences in their individual denaturation temperatures, which correlate with the observed temperature-dependent refinement of peak profiles by reversed-phase HPLC. This reversed-phase HPLC method in conjunction with other orthogonal analytical techniques (e.g. capillary gel electrophoresis, peptide mapping, ion exchange chromatography, etc.) is being used to characterize disulfide isomers in the development of therapeutic IgG2 antibodies.
本文报道了使用表面多孔颗粒填充的柱，快速反相 HPLC 分离重组人免疫球蛋白 G2（IgG2）二硫键异构体。在最佳条件下，通过组合使用高柱温（85°C）、高洗脱强度的流动相（如异丙醇）和高流速（1.5 mL/min），在 Poroshell™ 300SB-C8 柱上，10 min 内即可实现单克隆 IgG2 二硫键异构体的分离。对色谱富集的 IgG2 二硫键异构体的热力学稳定性分析表明，它们的个体变性温度存在差异，这与通过反相 HPLC 观察到的随温度变化的峰形细化相关。该反相 HPLC 方法与其他正交分析技术（例如毛细管凝胶电泳、肽图分析、离子交换色谱等）结合，用于在治疗性 IgG2 抗体的开发中表征二硫键异构体。
