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 Ca2+(300 mm×7.7 mm,8 μm)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10 μL,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 Ca2+ (300 mm×7.7 mm, 8 μm), 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 10 μL, 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.
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