首页|高浓度重链抗体可变区-Fc融合蛋白稳定性影响因素分析

高浓度重链抗体可变区-Fc融合蛋白稳定性影响因素分析

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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.
Analysis of factors affecting stability of high concentration VHH-Fc fusion protein
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.

Variable domain of heavy-chain antibody(VHH)Fc fragmentFusion proteinConformational stabilityColloidal stabilityAverage hydrodynamic diameterPost-translational modification

饶金瑞、王永红、沈智勇、赵黎明

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华东理工大学生物工程学院,上海 200237

上海臻格生物技术有限公司,上海 201318

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

国家重点研发计划

2022YFC2105100

2024

中国生物制品学杂志
中华预防医学会,长春生物制品研究所有限责任公司

中国生物制品学杂志

CSTPCD
影响因子：0.417
ISSN：1004-5503
年,卷(期)：2024.37(3)
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