平台化高通量pH梯度阳离子色谱法在单克隆抗体电荷异质性分析中的应用研究

目的：建立应用pH梯度分离的高通量、平台化高效液相阳离子交换色谱法，用于单克隆抗体电荷异质性分析。方法：利用不同pH的三羟甲基氨基甲烷、咪唑、哌嗪缓冲液组成pH梯度进行梯度洗脱，采用相同色谱柱、流动相和分离梯度对不同等电点的单抗进行分离。结果：用该法对美国药典收录的3种单抗-贝伐珠单抗、曲妥珠单抗、利妥昔单抗进行检测，并与对应各论中记载的盐梯度方法比较，结果显示3种单抗主峰与酸区、碱区的分离度均有改善，贝伐珠单抗分离度分别提高了44%和500%；曲妥珠单抗分离度分别提高了83%和233%；利妥昔单抗酸区分离度由无法给出提高到0.7，碱区分离度提高了10%。针对贝伐珠单抗的方法进行再次优化（提高40%通量）并开展方法学验证。结果显示方法专属性良好，高温破坏后可观察到酸区和碱区杂质均有趋势性增加；精密度良好，重复性实验中主峰保留时间RSD为0.12%，中间精密度实验中酸区、主峰、碱区百分比峰面积RSD均小于2%；准确度良好，酸区、主峰、碱区在5个不同的浓度级别中回收率总体平均值分别为100.4%，101.0%，101.4%；线性良好，蛋白含量0.8~1.2 mg·mL^-1范围内酸区、主峰、碱区峰面积均与样品浓度呈线性关系，主峰与碱区线性相关系数R²均大于0.999，酸区R²为0.988；耐用性良好，检测条件发生一定变化时，酸区、碱区和主峰的百分比峰面积测定RSD均小于3%。结论：建立的平台化pH梯度方法适用于不同单抗的电荷异质性分析，且具有良好的分离度，为单抗的研究和开发提供了一种通量更高的分离手段和分析方法。

Objective:To apply a high-throughput pH gradient-based ion-exchange chromatography method for the charge variants separation of several monoclonal antibodies.Methods:The buffer system consisted of tris,imidazole and piperazine.Each buffer was adjusted to different pH,respectively.A pH gradient method was used to separate MAbs with different pI values.Results:Three MAbs including bevacizumab,trastuzumab and rituximab, which were collected in USP Medicines Compendium,were separated and compared with their original salt-gradient method results.Results indicated that the resolution of acidic variants and basic variants were improved among all MAbs.In the case of bevacizumab,resolution improvements were 44% and 500%,respectively,and 83%,233% in trastuzumab,it is slightly better in the case of rituximab.The method was further improved for bevacizumab separation and validated.Separation time was shortened by 40%.The results of specificity test showed that variants were positively correlated with heating time,repeatability test showed that the RSD of retention time of main peak is 0.12%,inter-mediate test showed that the RSD of area percentage was less than 2%.Accuracy test showed that the overall recovery of variants and main peak were 100.4%,101.0%,and 101.4%,respectively.Linearity test showed that the linearity of variants and main peak was around 0.99.The method is robust if the chromatography condition deviate slightly,the RSD of variants and main peak were less than 3%.Conclusion:The pH gradient-based cation-exchange chromatography method is applicable for MAbs with different pI values,which provides a higher throughput and better separated analysis approach for monoclonal antibody study and development.