分析单克隆抗体电荷异质性的成像毛细管等电聚焦电泳技术平台方法的建立

目的  采用成像毛细管等电聚焦电泳技术（imaged capillary isoelectric focusing, iCIEF）建立分析单克隆抗体（单抗）电荷异质性的平台方法，并用不同亚型单抗（IgG1、IgG2、IgG4）确认该平台方法的适用性。方法  优化该平台方法的部分参数，包括两性电解质和阴极稳定剂体积、聚焦时间和尿素浓度。采用3种亚型单抗（IgG1、IgG2、IgG4）对该平台方法的专属性、精密度、线性、准确度和耐用性进行验证。结果   对样品（单抗）的处理条件为：3 mol/L尿素-0.5%甲基纤维素溶液70 μl、两性电解质（pH3~10）4 μl、阴极稳定剂（500 mmol/L 精氨酸）2 μl、等电点 6.14和9.99 Marker 各2 μl，最终完成0.2 mg/ml单抗（样品）的制备。检测参数：预聚焦1 500 V、1 min，聚焦3 000 V、8 min。该平台方法的专属性良好，制剂缓冲液对检测无干扰。重复检测6份平行样品以及不同分析员于不同时间检测12份样品各成分含量的相对标准偏差均符合规定的要求。单抗（样品）终浓度为0.1～0.3 mg/ml时，主要和酸性成分的线性决定系数（R2）≥0.99，碱性成分的线性R2≥0.98。该平台方法检测样品各成分的准确度为92～105%。耐用性实验设计结果表明，两性电解质（pH3~10）体积和毛细管批次对该平台方法有显著影响。结论  建立的iCIEF平台方法分离度较高，精密度、准确度和耐用性良好，为单抗制品的电荷异质性表征和质量控制提供了更有效的工具。

Development of iCIEF platform method for analysis of charge heterogeneity of monoclonal antibodies

Objective  To develope a platform method using imaged capillary isoelectric focusing (iCIEF) for measuring charge heterogeneity of monoclonal antibody (mAb) and validate its suitability by using different isoforms of mAbs (IgG1, IgG2, IgG4).  Methods   volumes of carrier ampholyte and cathode stabilizer, focusing time, and urea concentration were optimized in iCIEF method development. The specificity, precision, linearity, accuracy, and robustness of this method were validated using 3 isoforms of mAbs (IgG1, IgG2, IgG4). Results  Under following conditions: 70 μl 3 mol/L urea-0.5% methyl cellulose, 4 μl broad-range carrier ampholytes (pH3-10), 2 μl cathode stabilizer (500 mmol/L Arg), 2 μl each isoelectric point 6.14 and 9.99 Markers, 0.2 mg/ml mAb (sample) was prepared. The detection parameters were determined to be pre-focusing 1 min at 1 500 V and focusing 8 min at 3 000 V.  The specificity of the platform method was good, and formulation buffer had no interference for detection. Repeated testing of 6 parallel samples and testing 12 samples by different persons at different time both showed that, the relative standard deviations of all compositions met requirements. The linear coefficients of determination (R²) were ≥0.99 for main and acidic compositions in mAb and ≥0.98 for basic composition, when the final concentrations of mAbs (samples) were 0.1-0.3 mg/ml. The accuracies of the platform method for detecting  different composition contents in mAbs were 92%-105%. The robustness results of design of experiment showed that carrier ampholytes volume and capillary lot had significant impacts on this platform method.Conclusions  The iCIEF platform method for analysis of charge heterogeneity of different isoforms of mAbs is developed, which has high separation resolution and good precision, accuracy, and robustness. This method can provide a more effective tool for charge heterogeneity characterization and quality control of mAbs.