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

作　　者：饶金瑞 王永红[1] 沈智勇 赵黎明[1] RAO Jinrui;WANG Yonghong;SHEN Zhiyong;ZHAO Liming(School of Bioengineering,East China University of Science and Technology,Shanghai 200237,China;不详)

机构地区：[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

出　　处：《中国生物制品学杂志》2024年第3期273-279,共7页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2022YFC2105100)。

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,Met160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。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(T_(onset)),melting temperature(T_m)and aggregation onset temperature(T_(agg))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(k_D)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 

分 类 号：Q816[生物学—生物工程]