单抗制剂多维度稳定性分析  

Multi-Dimensional Stability Analysis of Monoclonal Antibodies

作　　者：郭莎[1] 张峰[1] 于传飞[1] 武刚[1] 崔永霏 王兰[1] GUO Sha;ZHANG Feng;YU Chuan-fei;WU Gang;CUI Yong-fei;WANG Lan(Division of Monoclonal Antibody Products,Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Product,National Institutes for Food and Drug Control,Beijing 102629,China)

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,北京102629

出　　处：《中国药学杂志》2021年第15期1241-1248,共8页Chinese Pharmaceutical Journal

基　　金：国家科技重大专项重大新药创制项目资助(2018ZX09101001-003);2020年国家药品标准提高项目课题资助(2020S11)。

摘　　要：目的单克隆抗体(单抗)的稳定性是抗体成药性研究中的重要内容,本研究拟探讨从单抗分子的结构稳定性、胶体稳定性及分子间弱相互作用等方面进行稳定性评价的意义方法本研究以曲妥珠、利妥昔及贝伐珠为例,从不同角度表征了这3种单抗的稳定性特点,展示了单抗分子稳定性评价的多种维度和思路首先,通过检测蛋白内源性荧光(intrinsic fluorescence,IF)技术测得熔解温度(melting temperature,Tm),对抗体分子的结构稳定性进行评估其次,采用静态光散射技术(static light scattering technique,SLS)预测抗体的胶体稳定性<随后,采用等温稳定性试验(60℃,48h)对样品的稳定性表现进行实时监测。最后,采用动态光散射技术(dynamic light scattering technique,DLS)从蛋白溶解状态下其自身分子间弱相互作用角度预测蛋白聚集倾向:结果IF技术检测发现3种抗体分子第1个熔解温度Tm1均在65℃以上,说明其结构均较稳定。SLS检测显示曲妥珠胶体稳定性最好,升温至95℃未检测到明显的聚集信号,而利妥昔和贝伐珠则在70℃左右出现明显聚集等温稳定性试验也显示了类似的结果。DLS技术检测显示,曲妥珠的第二维里系数(B_(22))和扩散相互作用系数(K_(D))均为正值(3.19×10^(-3)和65.46),预示长期胶体稳定性较好,利妥昔次之,而贝伐珠的B22和KD均为负值(-1.44×10^(-4)和6.46),提示蛋白分子间容易自缔合。结论本研究通过多种技术、从多种角度探讨了单抗药物稳定性评价的内容、层次和深度,为抗体分子筛选、制剂配方优化、抗体分子与制剂配方间的不同组合,以及稳定性监测等提供了借鉴和参考。OBJECTIVE To explore the significance of stability evaluation of monoclonal antibodies(mAbs), an important part of druggability of mAbs, from the aspects of structural stability, colloidal stability and weak intermolecular interaction. METHODS Taking trastuzumab, rituximab and bevacizumab as examples, the stability characteristics of the three mAbs were explored from different aspects, thus to demonstrate various dimensions of stability evaluation of mAbs. Firstly, the structural stability of antibody molecules was evaluated by detecting the melting temperature(Tm) through intrinsic fluorescence(IF). Secondly, static light scattering technique(SLS) was used to predict the colloidal stability of mAbs. Subsequently, isothermal stability tests(60 ℃, 48 h) were used to monitor the stability performance of the samples in real time. Lastly, dynamic light scattering technique(DLS) was used to predict protein aggregation tendency from the point of weak intermolecular interaction of dissolved proteins. RESULTS IF detection showed that the first melting temperature(Tm1) of the three mAbs were all above 65 ℃, indicating that their structures were stable. SLS detection showed that trastuzumab had the best colloidal stability since no obvious aggregation signal was detected at 95 ℃, while rituximab and bevacizumab showed obvious aggregation at about 70 ℃. The isothermal stability test also showed similar results. The application of DLS techniques showed that the second virial coefficient(B22) and diffusion interaction coefficient(KD) of trastuzumab were positive(3.19×10-3, 65.46), indicating better long term colloidal stability, rituximab was suboptimal, while the B22 and KD of bevacizumab were both negative(-1.44×10-4,-6.46), suggesting that the molecules were prone to be self-associated. CONCLUSION In this paper, the contents, levels and depth of the stability evaluation of mAbs are characterized by various techniques and discussed from multi-aspects, which provides reference for antibody molecular screening, form

关 键 词：单克隆抗体 结构稳定性 胶体稳定性 分子间弱相互作用 

分 类 号：R917[医药卫生—药物分析学]