利用分析超速离心技术探究异丙醇在抗体蛋白高聚体含量分析中的作用  

Exploration of the Role of Isopropanol in Antibody Aggregates by Analytical Ultracentrifugation

作　　者：李文 谢莎莎 韩文志 袁野 田橙 黄岗 曹晓林 LI Wen;XIE Shasha;HAN Wenzhi;YUAN Ye;TIAN Cheng;HUANG Gang;CAO Xiaolin(Analytical Science,Wuxi Biologics(Shanghai)Co.,Ltd.,Shanghai 200131,China)

机构地区：[1]上海药明生物技术有限公司分析科学部,上海200131

出　　处：《材料科学与工程学报》2023年第1期13-22,57,共11页Journal of Materials Science and Engineering

基　　金：上海药明生物生物物理平台研发资助项目(I001185)。

摘　　要：蛋白抗体聚集是影响药物质量的原因之一。体积排阻色谱(SEC)技术是蛋白质药物聚集体表征的常用方法,但由于制剂配方与SEC流动相较难完全一致,会引起蛋白单体非共价聚集分布,影响聚集体测定结果。本研究采用分析超速离心-沉降速率技术与体积排阻-多角度静态光散射技术以及动态光散射技术相结合的方法,研究了单克隆抗体及抗体偶联药物在添加10%异丙醇的体积排阻色谱流动相体系中单体和二聚体百分含量的变化规律。结果表明,异丙醇有助于消除单体间相互作用,降低非共价二聚体形成,并起到稳定蛋白单体形态及蛋白构象的作用,为体积排阻色谱法分析易聚集蛋白提供了更加准确的结果。在高盐添加10%异丙醇的流动相体系中,分析超速离心分析得到的分子质量、单体纯度、蛋白摩擦系数、水合半径、轴长比等水力学参数均可达到与制剂缓冲液一致的结果,说明流动相中添加低比例的异丙醇可以得到更高的单体含量,与实际治疗的制剂状态具有等同意义。本研究表明,将体积排阻色谱-多角度静态光散射与分析超速离心技术相结合可以有效地分析抗体蛋白的纯度和聚集形态,是抗体蛋白质量分析及制剂筛选的可靠手段。Protein antibody aggregation severely affects the quality of drugs.Size exclusion chromatography(SEC)is a common method to analyze the state of aggregates of protein drugs.However,it is difficult for the formulation to be completely consistent with the SEC mobile phase,which may cause nonprotein monomers.Covalent aggregation distribution affects the result of aggregate determination.Utilizing the combined methods of analytical ultracentrifugation-sedimentation velocity(AUC-SV),size exclusion-static light scattering(SEC-MALS)and dynamic light scattering(DLS),the changes of monomer and dimer contents for monoclonal antibody(mAb)and antibody-drug conjugate(ADC)were studied in the mobile phase buffer with 10%isopropanol addition.The results show that isopropanol can help to eliminate the interactions between monomers,reduce the formation of noncovalent bonded dimer,and play the role in stabilizing the protein monomers and conformations,providing more accurate results for SEC analysis of aggregation-prone proteins.In analytical ultracentrifugation study,the protein molecular weight,monomer percentage,friction coefficient,Stokes radius,axial ratio and other hydrodynamic parameters obtained in the mobile phase buffer with high salt concentration adding 10%isopropanol were consistent with those in formulation buffer,indicating a similar practical effect with that of real drug formulation.This study reveals that the combination of SEC-MALS and AUC can be effectively applied in analyzing the purity and aggregation of antibody protein.It is one of reliable methods for antibody protein quality analysis and formulation buffer screening.

关 键 词：分析超速离心技术 体积排阻色谱 多角度静态光散射 抗体偶联药物 异丙醇 制剂缓冲液 

分 类 号：Q67[生物学—生物物理学]