糖基化修饰对重组CTLA4-Ig稳定性影响的研究

目的：探讨糖基化修饰对细胞毒性T淋巴细胞相关抗原4抗体融合蛋白（CTLA4-Ig）融合蛋白稳定性的影响。方法：应用多种糖苷酶N-糖苷酶F（PNGase F）、内切糖苷酶F2（EndoF2）、唾液酸酶（neuraminidase）和O-糖苷酶（O-glycosidase）对样品进行处理。通过分子排阻色谱（SEC）测定不同酶切时间处理后样品纯度变化；应用差示扫描荧光（DSF）测定样品整体热力学参数的变化；应用差示扫描量热（DSC）测定样品各结构域热力学参数变化；在45℃条件下加热，分别于0、1、3、5、7、9、11和12周取样，通过SEC测定样品纯度的变化。结果：在N-糖苷酶F、内切糖苷酶F2和唾液酸酶分别处理过程中，CTLA4-Ig的聚体含量逐渐增加，而O-糖苷酶酶切过程中，聚体虽无明显变化，但碎片含量逐渐增加；经DSF测定，N-糖苷酶F和内切糖苷酶F2处理后解链温度（T_m）均明显下降，而唾液酸酶和O-糖苷酶处理后T_m无明显变化；经DSC测定，N-糖苷酶F和内切糖苷酶F2处理后T_m1下降，T_m2不变，T_m3上升，而唾液酸酶处理后T_m1和T_m3都略微有所上升，T_m2基本不变，O-糖苷酶处理后T_m值均变化；加速试验过程中，N-糖苷酶F和唾液酸酶处理样品的聚体上升，内切糖苷酶F2处理样品的的聚体不变；O-糖苷酶处理样品的的聚体不变，而碎片偏高。结论：通过多种分析手段研究发现，糖基化修饰与CTLA4-Ig的稳定性密切相关。

Objective:To investigate the effect of glycosylation modification on the stability of cytotoxic T-lymphocyte- associated antigen 4-immunoglobulin(CTLA4-Ig). Methods:The samples were treated with various glycosidase enzymes (PNGase F,EndoF2,neuraminidase and O-glycosidase). the changes of sample purity were determined by molecularsize exclusion chromatography(SEC),tested overall thermodynamic parameters by differential scanning fluoroscopy(DSF),and analyzed by differential scanning calorimetry(DSC) for each domain thermodynamic parameters. Heated at 45,the samples were determined by SEC for purity analysis at 0,1,3,5,7,9,11 and 12 weeks,respectively. Results:During the treatment of PNGase F,EndoF2 and neuraminidase,respectively,the content of CTLA4-Ig polymer increased gradually. The dimmer had no obvious variation in O-glycosidase digestion process,but the debris content gradually increased. DSF analysis showed that the melting temperature(T_m) of samples treated with PNGase F and EndoF2 were significantly decreased,while that of treated with neuraminidase and and O-glycosidase were no significant changes. DSC analaysis showed that T_m1 decreased,T_m2 remained unchanges and T_m3 increased after PNGase F and EndoF2 treatment, T_m1 and T_m3 slightly increased and T_m2 remained unchanged after neuraminidase treatment,and T_m values changed after O-glycosidase treatment. In the accelerated experiment,the dimers of PNGase F and neuraminidase treated samples increased,it was unchanged to EndoF2 treated samples. To O-glycosidase treated samples,the dimer unchanged,while the fragments were slightly higher. Conclusion:It was found that glycosylation modification was closely related to the stability of CTLA4-Ig by various analytical methods.

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