高浓度重链抗体可变区-Fc融合蛋白稳定性影响因素分析
Analysis of factors affecting stability of high concentration VHH-Fc fusion protein
饶金瑞 1王永红 1沈智勇 2赵黎明1
作者信息
- 1. 华东理工大学生物工程学院,上海 200237
- 2. 上海臻格生物技术有限公司,上海 201318
- 折叠
摘要
目的 分析高浓度重链抗体可变区-Fc(variable domain of heavy-chain antibody-Fc,VHH-Fc)融合蛋白稳定性的影响因素.方法 设置振摇、光照、40 ℃高温3组强制降解试验,通过差式扫描荧光法、动态光散射法(dynamic light scattering,DLS)和超高效液相色谱-质谱(ultra performance liquid chromatography-mass spectrometry,UPLC-MS)法检测3种强制降解条件对高浓度VHH-Fc融合蛋白构象稳定性、胶体稳定性、平均水化动力学直径及翻译后修饰的影响.结果 光照条件下,高浓度VHH-Fc融合蛋白的构象展开起始温度(onset temperature of unfolding,Tonset)、熔解温度(melting temperature,Tm)和起始聚集温度(aggregation onset temperature,Tagg)降低幅度最大,Met 160和 Met266处的氧化比例大幅增加;振摇条件下,扩散相互作用系数(diffusion interaction parameter,kD)和平均水化动力学直径变化量最大.结论 光照会显著降低高浓度VHH-Fc融合蛋白的构象稳定性并诱导甲硫氨酸氧化,振摇对其胶体稳定性影响最为显著且会促进聚集.
Abstract
Objective To explore the factors affecting the stability of high concentration variable domain of heavy-chain antibody-Fc(VHH-Fc)fusion protein.Methods Three groups of forced degradation experiments,shaking,light and 40 ℃ high temperature were set up.Differential scanning fluorimetry,dynamic light scattering(DLS)and ultra performance liquid chromatography-mass spectrometry(UPLC-MS)were used to detect the effects of the three forced degradation conditions on the conformational stability,colloidal stability,average hydrodynamic diameter and post-translational modifications of high concentration VHH-Fc fusion protein.Results Under the light condition,the onset temperature of unfolding(Tonset),melting temperature(Tm)and aggregation onset temperature(Tags)of high concentration VHH-Fc fusion protein decreased the most,and the oxidation ratio of Met160 and Met266 increased significantly.Under the condition of shaking,the variation of the diffusion interaction parameter(kD)and the average hydrodynamic diameter was the largest.Conclusion Light can significantly reduce the conformational stability of high concentration VHH-Fc fusion protein and induce methionine oxidation.Shaking has the most significant effect on its colloidal stability and promotes aggregation.
关键词
重链抗体可变区/Fc片段/融合蛋白/构象稳定性/胶体稳定性/平均水化动力学直径/翻译后修饰Key words
Variable domain of heavy-chain antibody(VHH)/Fc fragment/Fusion protein/Conformational stability/Colloidal stability/Average hydrodynamic diameter/Post-translational modification引用本文复制引用
基金项目
国家重点研发计划(2022YFC2105100)
出版年
2024
NETL
NSTL
万方数据