多元醇对全人源抗VEGF_(165)单克隆抗体构象稳定性及胶体稳定性的影响  

Effects of Polyols on Human Anti-VEGF_(165) Monoclonal Antibody Conformational Stability and Colloidal Stability

作　　者：高飞[1] 刘煜[1] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《药物生物技术》2016年第3期208-212,共5页Pharmaceutical Biotechnology

基　　金：国家"重大新药创制"科技重大专项资助项目(No.2013ZX09301303-004);江苏高校品牌专业建设工程资助项目(No.PPZY2015A057);江苏省重点高校学术程序开发资助项目(PAPD)

摘　　要：通过研究4种多元醇(海藻糖、甘露醇、山梨糖和蔗糖)对全人源抗VEGF165单克隆抗体(HVAB)构象及胶体稳定性的影响,筛选出最优的HVAB稳定剂并探讨多元醇稳定抗体的作用机制。该研究运用差示荧光扫描法(Differential scanning fluorimetry,DSF)判断多元醇对抗体热稳定性的影响;采用圆二色光谱(Circular dichroism,CD)和二阶导数色氨酸荧光光谱探讨多元醇对抗体构象稳定性的影响;聚乙二醇诱导抗体发生液-液相分离现象判断多元醇对抗体胶体稳定性的影响。结果表明4种多元醇均能提高抗体的热稳定性及胶体稳定性;多元醇作为辅料添加到溶液中会引起抗体构象发生变化,圆二色谱结果显示HVAB二级结构发生变化,但仍然以β-折叠为主;海藻糖和甘露醇引起HVAB发生去折叠,并未引起抗体发生聚集或片段化,同时提高了抗体的胶体稳定性;山梨糖提高抗体Tm值效果最明显,在稳定抗体胶体结构方面也表现突出,且作为添加剂对抗体二级结构影响最小。说明抗体的构象稳定性和胶体稳定性相互影响;山梨糖可作为HVAB的最优稳定剂。To determine the effects of four polyols （trehalose, mannitol, sorbitol and sucrose） on human anti-VEGF165 monoclonal antibody （HVAB） stability and screening optimal formulation stabilizers for HVAB, and to explore the mechanism of how polyols make antibody stable, differential scanning fluorimetry （DSF） was used to study thermal stability of HVAB in various formulations and screening suitable thermal accelerated temperature. SEC-HPLC was used to detect the contents change of antibody aggregates, monomers and fragments during thermal acceleration test. Circular dichroism （CD） and second derivative tryptophan fluorescence spectroscopy were used to study conformational stability of HVAB. Polyethylene glycol（PEG） induced liquid-liquid phase separation （LLPS） phenomenon to study colloidal stability of HVAB. The results showed that the thermal stability and colloidal stability of the antibody could be improved by the four polyols,especially by sorbitol. Polyols as stabilizer added to the solution causes conformational changes in antibody. Circular dichroism results showed that, compared with the control group, the second structure of HVAB in four kinds of the polyol grouphas changed, but remains containing mainly β-stand. Trehalose and mannitol cause HVAB tertiary structure changes, but did not cause antibody aggregation or fragmentation and it may be due to the polyol enhance of the colloidal stability of the antibody. These results indicated that sorbitol can improve Tm of HVAB the most, also can enhance the colloidal stability the most and minimal effects on the secondary structure of HVAB. That is why sorbitol can be used for the optimal stabilizer.

关 键 词：多元醇 单克隆抗体 构象稳定性 胶体稳定性 圆二色谱 差示荧光扫描 液-液相分离 

分 类 号：Q81[生物学—生物工程]