摘要
目的建立治疗性单克隆抗体(单抗)SIBP-A电荷异构体全柱成像毛细管等电聚焦电泳(imaging capillary isoelectric focusing electrophoresis, icIEF)的测定方法, 并进行验证。方法针对尿素浓度、两性电解质范围及浓度和聚焦时间对icIEF方法进行条件优化, 并应用优化条件对该单抗电荷异构体进行分析, 验证专属性、稳定性指示性、重复性、中间精密度、线性与范围、准确度、定量限及处理后样品的稳定性。结果优化后icIEF条件:3 mol/L尿素、1% MC胶、3%两性电解质载体Pharmalyte 3-10与Pharmalyte 8-10.5按1∶1混合, 电压1 kV、预聚焦1 min, 电压3 kV、聚焦分离6 min。专属性实验中, 空白组在样品出峰处无明显干扰峰, 对照组正常出峰且电荷异构体正常;稳定性指示性实验中, 可以检测出40 ℃放置7 d的样品正常出峰而且电荷异构体发生显著变化, 与对照样品明显不同;重复性实验中, 酸性峰、主峰及碱性峰相对百分含量的相对标准偏差(relative standard deviation, RSD)分别为0.48%、0.00%和4.03%, 主峰等电点RSD为0.012%;中间精密度实验中, 酸性峰、主峰及碱性峰相对百分含量的RSD分别为0.89%、0.45%和6.14%, 主峰等电点RSD为0.072%;在蛋白终浓度0.50~1.50 mg/ml范围内具有良好的线性, 线性决定系数为0.996 8, 且回收率在95.03%~104.35%内;该方法的定量限为0.003 0 mg/ml;10 ℃条件下样品酸性峰、主峰、碱性峰相对百分含量的RSD分别为0.56%、0.00%和8.61%, 主峰等电点RSD为0.056%;室温条件下3 h酸性峰电荷异构体开始发生明显变化。结论研究建立的检测方法可以有效检测单抗SIBP-A的电荷异构体及等电点, 专属性、稳定性指示性、重复性、中间精密度、线性与范围、准确度、定量限和10 ℃条件下样品的稳定性均良好, 可用于该抗体的放行检测及稳定性检测。
ObjectiveTo establish and validate an imaging capillary isoelectric focusing electrophoresis(icIEF)method for the detection of charge variants in the therapeutic monoclonal antibody SIBP-A.MethodsThe icIEF conditions were first optimized by adjusting urea concentration,ampholyte range,ampholyte concentration and focusing time.The optimized method was then applied to analyze the charge variants of SIBP-A,and its specificity,stability indicating property,repeatability,intermediate precision,linearity and range,accuracy,limit of quantification and stability of processed samples were verified.ResultsThe optimized icIEF conditions included 3 mol/L urea,1%MC gel,and a 1∶1 mixture of two ampholytes(Pharmalyte 3-10 and Pharmalyte 8-10.5)as carriers,1 kV prefocusing voltage with 1 min prefocusing time,and 3 kV focusing voltage with 6 min focusing time.In the specificity experiment,the blank group registered no significant interference peak at the sample peak while the control group exhibited normal peaks and charge variants.In the stability indicating experiment at 40℃for 7 d,the sample showed normal peaks but with significant changes in charge isoforms,which were distinct from the control sample.The repeatability experiment demonstrated relative standard deviation(RSD)values of 0.48%,0.00%,and 4.03%for the acidic,main,and basic peak relative percentage contents,respectively,and an RSD of 0.012%for the main peak’s isoelectric point.Precision experiments showed RSD values of 0.89%,0.45%,and 6.14%for the acidic,main,and basic peak relative percentage contents,respectively,with an RSD of 0.072%for the main peak’s isoelectric point.In the protein range of 0.50-1.50 mg/ml,the coefficient of determination of the linear regression cure of the total peak area was 0.9968 with good linearity,and the recovery rate of each protein concentration were 95.03%-104.35%.The limit of quantitation was 0.0030 mg/ml.The sample of stability experiments showed RSD values of 0.56%,0.00%,and 8.61%for the acidic,main,and basic peak relative
作者
张琳
陈慧聪
蔡丽星
蒋晴怡
梁红远
张坤明
卢逸仁
沈培玉
马雷钧
邱建华
Zhang Lin;Chen Huicong;Cai Lixing;Jiang Qingyi;Liang Hongyuan;Zhang Kunming;Lu Yiren;Shen Peiyu;Ma Leijun;Qiu Jianhua(No.1 Research Laboratory,Shanghai Institute of Biological Products Co.,Ltd.,Shanghai 200051,China;Quality Control Department,Shanghai Institute of Biological Products Co.,Ltd.,Shanghai 201403,China)
出处
《国际生物制品学杂志》
CAS
2024年第3期156-163,共8页
International Journal of Biologicals
关键词
治疗性单克隆抗体
全柱成像毛细管等电聚焦电泳
电荷异构体
方法学验证
Therapeutic monoclonal antibody
Imaging capillary isoelectric focusing electrophoresis
Charge variants
Method validation
