摘要
目的:建立定量测定溶剂中低浓度重组人源化抗BCMA/CD3双特异性抗体药物浓度的分子体积排阻高效液相色谱法(SEC-HPLC法)。方法:采用TSKgel G3000SWxl(7.8 mm×300 mm,5μm)体积排阻色谱柱,高效液相紫外检测器在214 nm处测定抗体药物吸收度。对SEC-HPLC法进行专属性、系统残留、线性、精密度、稳定性方法学验证,并使用该方法和紫外-可见分光光度法测定样品浓度,进行准确度分析。结果:SEC-HPLC法对抗体药物浓度的测定无干扰;在0.01~0.32 mg·mL^-1浓度范围内,SECHPLC法线性良好(相关系数R2=1.000);不同实验者不同时间使用该方法可准确测定抗体药物浓度,RSD均≤2.0%,回收率均处于90.0%~110.0%之间;-80℃反复冻融和2~8℃存放24 h后,SEC-HPLC法仍能准确测定抗体药物浓度,稳定性良好(RSD≤2.0%)。SEC-HPLC法能准确测定样品浓度,RSD≤2.0%,回收率均处于90.0%~110.0%之间,而紫外-可见分光光度法不能准确测定低浓度(0.01 mg·m L^-1)样品的浓度,RSD为5.7%,回收率为131.6%。结论:SEC-HPLC测定浓度法准确性和精密度良好,能准确测定溶媒剂中0.01~0.32 mg·mL^-1范围内抗体药物浓度,能避免紫外-可见分光光度法测定0.01 mg·mL^-1样品不准确的局限性,可作为测定低浓度重组人源化抗BCMA/CD3双特异性抗体药物浓度的有效方法之一。
Objective:To establish the quantitative determination method of low concentration recombinant humanized anti-BCMA/CD3 bispecific antibody drug by SEC-HPLC in solvents. Methods:An SEC-HPLC method was developed to determine concentration on TSKgel G3000 SWxl(7.8 mm×300 mm,5 μm)size exclusion chromatography column with UV detection at 214 nm. The method was verified for specificity,system residue,linearity,precision and stability according to the method validation requirements. Concentration of sample was determined with SEC-HPLC and UV-Vis(ultraviolet and visible)spectrophotometric to mensurate recovery rate. Results:The antibody drug concentration was determined with SECHPLC without interference. The linear correlation was good within the concentration range of 0.01-0.32 mg·mL^-1(R2=1.00) and the recovery rates were all 90.0%-110.0%. Different experimenters using this method at different times could accurately determine the concentration of antibody drug(RSD ≤ 2.0%).The recovery rates were between 90.0%-110.0%. Concentration of antibody drug which repeated freezing and thawing at-80 ℃ and storaged at 2-8 ℃ for 24 h was still accurately determined with SEC-HPLC(RSD ≤ 2.0%). SEC-HPLC method could accurately determine the concentration of the sample(RSD ≤ 2.0%).UV-vis spectrophotometry had poor accuracy for determining low concentration(0.01 mg·mL^-1)(RSD = 5.7%). The recovery rate was 131.6%. Conclusion:The proposed SEC-HPLC method is simple and accurate.It can accurately determine the concentration of antibody drug in the range of 0.01-0.32 mg·mL^-1 and can effectively avoid the shortcomings issued of UV-vis spectrophotometry which has poor accuracy for determining 0.01 mg·mL^-1 concentration. SEC-HPLC can be widely applied as an effectively method for quantitative analysis of low concentration recombinant humanized anti-BCMA/CD3 bispecific antibody drug.
作者
廉开礼
崔岩
彭伟
曹宇
刘忠
张贵民
赵丽丽
LIAN Kai-li;CUI Yan;PENG Wei;CAO Yu;LIU Zhong;ZHANG Gui-min;ZHAO Li-li(National Engineering Lab for High Efficiency Expression of the Mammalian Cells,Shandong New Time Pharmaceutical Co.,Ltd.,Linyi 273400,China)
出处
《药物分析杂志》
CAS
CSCD
北大核心
2020年第8期1365-1370,共6页
Chinese Journal of Pharmaceutical Analysis
