高效液相色谱-电喷雾法测定单克隆抗体药物中海藻糖的含量

Development of an HPLC-CAD method to determine trehalose in therapeutic monoclonal antibody

目的:建立采用高效液相色谱-电喷雾检测器(high performance liquid chromatography-charged aerosol detector,HPLC-CAD)测定单克隆抗体类药物中海藻糖含量的方法。方法:采用HyperREZ XP Carbohydrate Ca_(2)*(300 mm×7.7 mm,8μm)色谱柱,以超纯水为流动相,流速0.8 mL·min^(-1),柱温60℃,检测时间15 min,进样量10μL,CAD雾化室温度为35℃,Filter为5.0。结果:海藻糖对照品50%、80%、100%、120%及150%浓度水平验证溶液的回收率在90.8%~105.5%范围内,3次进样的峰面积RSD均小于10%,100%浓度水平验证溶液6次进样海藻糖含量测定值的RSD为2.7%;3批样品每批平行配制3份,测定结果均值分别为1.054、1.041及0.995 mg·mL^(-1),RSD分别为3.4%、3.1%及6.1%。结论:该方法简便、快速、灵敏,可用于准确测定单克隆抗体药物单克隆抗体药物中海藻糖的含量。

Objective:To establish a method for the determination of trehalose in monoclonal antibodies by high performance liquid chromatography-charged aerosol detector(HPLC-CAD).Methods:The chromatographic column was HyperREZ XP Carbohydrate Ca_(2)*(300 mm×7.7 mm,8μm),the mobile phase was ultra pure water,the flow rate was 0.8 mL·min^(-1),the column temperature was 60℃,the detection time was 15 min,the injection amount was 10μL,the CAD atomizing chamber temperature was 35℃,and the filter was 5.0.Results:The recoveries of validation solutions at 50%,80%,100%,120% and 150% concentrations were in the range of 90.8%-105.5%.The RSDs of peak areas were all below 10%.The RSD of measured value of validation solution at 100%concentration level was 2.7%after 6 times injection.Three batches of samples were prepared,each batch was prepared three times in parallel,and the mean values of measured results were 1.054,1.041 and 0.995 mg·mL^(-1),with RSDs of 3.4%,3.1%and 6.1%,respectively.Conclusion:This method is simple,rapid and sensitive,and can be used to accurately determine the trehalose content in monoclonal antibodies.