HPLC法同时测定单抗药物中海藻糖、甘露醇和山梨醇的含量  

Simultaneous determination of trehalose,mannitol and sorbitol in monoclonal antibody drugs by HPLC method

作　　者：崔春博 杜加亮[1] 武刚[1] 李萌[1] 徐刚领 高旭 俞小娟 倪永波 于传飞[1] CUI Chunbo;DU Jialiang;WU Gang;LI Meng;XU Gangling;GAO Xu;YU Xiaojuan;NI Yongbo;YU Chuanfei(Division of Monoclonal Antibody Products,National Institutes for Food and Drug Control,Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,Key Laboratory of the National Medical Products Administration for Research on Quality Study and Evaluation of Biological Products,Beijing 102629,China;Beijing Institute for Drug Control)

机构地区：[1]中国食品药品检定研究院单克隆抗体产品室,卫生部生物技术产品检定及标准化重点实验室,国家药品监督管理局生物制品质量研究与评价重点实验室,北京102629 [2]北京市药品检验研究院

出　　处：《山西医科大学学报》2024年第11期1419-1428,共10页Journal of Shanxi Medical University

基　　金：国家重点研发计划项目(2021YFF0600804)。

摘　　要：目的 建立同时检测单抗药物中海藻糖、甘露醇和山梨醇含量的方法。方法 采用伯乐碳水化合物快速分析高效液相色谱柱(7.8 mm×100 mm,9μm)进行海藻糖、甘露醇和山梨醇含量的测定,以超纯水为流动相,流速为0.5 mL/min,柱温为65℃,检测器为示差折光检测器。结果 该方法专属性良好,在海藻糖、甘露醇和山梨醇的保留时间范围内,无分析溶剂的干扰。海藻糖、甘露醇和山梨醇含量在0.16~1.60 mg/mL范围内,进样量与峰面积均呈良好的线性关系(R^(2)>0.99)。对单抗药物同时加标0.4 mg/mL海藻糖、0.4 mg/mL甘露醇和0.4 mg/mL山梨醇的样品进行6次独立测定,其海藻糖、甘露醇和山梨醇含量的变异系数分别为1.52%,2.54%和2.33%;对单抗药物同时加标0.8 mg/mL海藻糖、0.8 mg/mL甘露醇和0.8 mg/mL山梨醇的样品进行6次独立测定,其海藻糖、甘露醇和山梨醇含量的变异系数分别为0.69%,0.27%和0.18%;对单抗药物同时加标1.2 mg/mL海藻糖、1.2 mg/mL甘露醇和1.2 mg/mL山梨醇的样品进行6次独立测定,其海藻糖、甘露醇和山梨醇含量的变异系数分别为3.45%,6.55%和2.47%;以上结果均符合重复性的可接受标准(≤10%)。两名实验人员分别在3个工作日对上述3种加标溶液的海藻糖、甘露醇和山梨醇含量的样品共进行18次测定,其海藻糖含量的变异系数分别为2.08%,2.82%和10.87%;甘露醇含量的变异系数分别为2.24%,1.99%和2.53%;山梨醇含量的变异系数分别为2.54%,1.62%和1.32%;以上结果符合中间精密度的可接受标准(≤15%)。上述3种加标溶液的回收率均在97.88%~101.39%之间,符合准确度的验证要求(90%~110%);上述3种加标溶液在(5±3)℃密封瓶条件下在自动进样器中暂存24 h后海藻糖、甘露醇和山梨醇含量的平均回收率均在93.24%~101.49%之间,符合耐用性的可接受标准(90%~110%)。结论 成功建立了同时检测单抗药物中海藻糖、甘露醇和山梨醇含量的高�Objective To establish a method for simultaneously detecting trehalose,mannitol and sorbitol contents in monoclonal an-tibody drugs.Methods The contents of trehalose,mannitol and sorbitol were determined on a Bio-Rad HPLC fast carbohydrate analysis column(7.8 mm×100 mm,9µm),with ultrapure water as the mobile phase at a flow rate of 0.5 mL/min.The column tempera-ture was 65℃and the refractive index detector was adopted.Results The method had good specificity,and there was no interfer-ence from the analyzed solvents in the range of retention time of trehalose,mannitol and sorbitol.The contents of trehalose,mannitol and sorbitol showed good linearity between the injection weight and the peak area in the range of 0.16-1.60 mg/mL(R^(2)˃0.99).Six inde-pendent assays were performed on samples simultaneously spiked with 0.4 mg/mL trehalose,0.4 mg/mL mannitol and 0.4 mg/mL sorbi-tol for monoclonal antibody drugs,and the coefficients of variation for trehalose,mannitol and sorbitol contents were 1.52%,2.54%and 2.33%,respectively.Six independent assays were performed on samples simultaneously spiked with 0.8 mg/mL trehalose,0.8 mg/mL mannitol and 0.8 mg/mL sorbitol for monoclonal antibody drugs,and the coefficients of variation for trehalose,mannitol and sorbitol contents were 0.69%,0.27%and 0.18%,respectively.Six independent assays were performed on samples simultaneously spiked with 1.2 mg/mL trehalose,1.2 mg/mL mannitol and 1.2 mg/mL sorbitol for monoclonal antibody drugs,and the coefficients of variation for trehalose,mannitol and sorbitol contents were 3.45%,6.55%and 2.47%,respectively.All results met the acceptable criteria for re-peatability(≤10%).After the trehalose,mannitol and sorbitol contents in the three spiked solutions mentioned above were assayed 18 times by two staffs over three working days,the respective coefficients for trehalose contents were 2.08%,2.82%and 10.87%,the re-spective coefficients for mannitol contents were 2.24%,1.99%and 2.53%,and the respective coefficients for sorbitol contents wer

关 键 词：单克隆抗体药物 海藻糖 甘露醇 山梨醇 高效液相色谱法 辅料 质量控制 

分 类 号：R927[医药卫生—药学]