玻璃内包材界面修饰对机械应力诱导的单克隆抗体聚集体形成的影响

doi:10.11949/0438-1157.20230201

摘要/Abstract

摘要：

利用十八烷基三氯硅烷（octadecyltrichlorosilane，OTS）对中硼硅玻璃管制注射剂瓶内表面进行化学改性，使用接触角仪（水接触角从50°±1°增加至90°±2°）、原子力显微镜（表面粗糙度从0.448±0.086增加至1.282±0.117）、红外（出现—CH₂—和—CH₃特征峰）对表面进行验证，成功制备了具有疏水性能的玻璃表面。给玻璃瓶中的单克隆抗体施加机械应力，使用微流成像技术、尺寸排阻-高效液相色谱法（size exclusion-high performance liquid chromatography，SE-HPLC）、外源荧光对单抗制剂的稳定性进行全面表征。结果表明，OTS处理的疏水性界面可以减少机械应力诱导的蛋白质聚集体的产生。由于疏水表面与蛋白质强的疏水相互作用，OTS处理的疏水表面可有效抑制机械应力诱导的抗体分子的聚集。

关键词: 表面化学, 内包材, 吸附, 抗体, 聚集

Abstract:

The inner surface of middle borosilicate glass tubing was modified by octadecyltrichlorosilane (OTS). The contact angles of modified surfaces which were measured by contact angle goniometer increased from 50°±1° to 90°±2°. The surface roughness which was measured by atomic force microscope increased from 0.448±0.086 to 1.282±0.117, and the result of Fourier transform infrared spectroscopy (FTIR) indicated that the OTS was successfully coated on the glass surfaces. The monoclonal antibody in glass containers were subjected to mechanical stress. Microflow imaging, size exclusion-high performance liquid chromatography (SE-HPLC) and extrinsic fluorescence dye were used to characterize the stability of monoclonal antibody. The results showed that the OTS-treated hydrophobic interface could reduce the generation of mechanical stress-induced protein aggregates. Due to the strong hydrophobic interaction between hydrophobic surface and protein, the hydrophobic surface could effectively reduce the aggregation of antibody molecules induced by mechanical stress.

Key words: surface chemistry, primary container, absorption, antibody, aggregation

中图分类号:

TQ 464.9

王新悦, 王俊杰, 曹思贤, 王翠, 李灵坤, 吴宏宇, 韩静, 吴昊. 玻璃内包材界面修饰对机械应力诱导的单克隆抗体聚集体形成的影响[J]. 化工学报, 2023, 74(6): 2580-2588.

Xinyue WANG, Junjie WANG, Sixian CAO, Cui WANG, Lingkun LI, Hongyu WU, Jing HAN, Hao WU. Effect of glass primary container surface modification on monoclonal antibody aggregates induced by mechanical stress[J]. CIESC Journal, 2023, 74(6): 2580-2588.

图/表 10

图1 OTS在玻璃表面化学处理的流程图

Fig.1 Flow chart of chemical reaction of OTS on glass surface

图2 接触角检测结果

Fig.2 Results of water contact angle

图3 玻璃表面的FTIR谱图a—未处理的玻璃表面；b—0.17 μmol/L OTS处理的玻璃表面；c—0.85 μmol/L OTS处理的玻璃表面；d—3.4 μmol/L OTS处理的玻璃表面；e—17 μmol/L OTS处理的玻璃表面

Fig.3 FTIR spectrum of glass surfaces

图4 原子力显微镜表面形貌三维图

Fig.4 Three-dimensional surface topography of AFM

图5 IgG2在玻璃表面的Langmuir吸附模型a—未处理的玻璃表面;b—0.17 μmol/L OTS处理的玻璃表面;c—0.85 μmol/L OTS处理的玻璃表面;d—3.4 μmol/L OTS处理的玻璃表面;e—17 μmol/L OTS处理的玻璃表面

Fig.5 Langmuir adsorption model of IgG2 on glass surfaces

图6 在未处理、0.17 μmol/L OTS和17 μmol/L OTS处理的玻璃瓶中，经过机械应力处理的 IgG2产生的蛋白质聚集体

Fig.6 IgG2 protein aggregates produced by mechanically stressed in untreated and OTS glass vials

图7 机械应力处理后的单抗溶液产生的亚可见颗粒的粒径分布

Fig.7 Size distribution of subvisible particles in monoclonal antibody after mechanical stress treatment

图8 在未处理和OTS处理的小瓶中经过机械应力处理后的单抗溶液产生的亚可见颗粒的形态

Fig.8 Morphology of subvisible particles in monoclonal antibody solution after mechanical stress treatment in untreated glass vials and glass vials coated by 17 μmol/L OTS

图9 IgG2亚可见颗粒的粒径和圆度之间的等高线图

Fig.9 Contour plots of IgG2 subvisible particle size and circularity

图10 可溶性IgG2的SE-HPLC谱图a—未受应力的IgG2; b—未处理的玻璃瓶中经过应力处理的IgG2;c—0.17 μmol/L OTS处理的玻璃瓶中应力处理的IgG2;d—17 μmol/L OTS处理的玻璃瓶中应力处理的IgG2

Fig.10 SE-HPLC spectrum of soluble IgG2

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