靶向PD-L1/TGF-β双特异性抗体N538位点糖基化差异研究

Differences of glycosylation at N538 site of targeted PD-L1/TGF-βbispecific antibody

目的:研究N538位点糖基化差异对靶向PD-L1/TGF-β双特异性抗体高级结构及体外活性的影响,为产品质量控制策略的制定提供参考依据。方法:采用疏水层析法(HIC)分离得到具有糖基化差异的样品,并通过圆二色性(CD)、差示扫描量热法(DSC)等技术对其结构进行表征,通过表面等离子共振技术(SPR)研究其对TGF-β1的亲和力,采用酶联免疫吸附测定法(ELISA)以及报告基因法(RGA)进行体外活性研究。结果:研究得到了具有糖基化差异的样品,结果证明N538位点糖基化对双特异性抗体的圆二色性、热稳定性、亲和力以及TGF-β1抗原结合活性和细胞内结合TGF-β1的相对活性均无明显影响。结论:N538位点糖基化并不影响靶向PD-L1/TGF-β双特异性抗体产品的质量,因此在生产过程中可不对N538位点糖基化程度进行控制。

Objective:To study the effect of glycosylation at N538 site on the advanced structure and in vitro activity of the targeted PD-L1/TGF-βbispecific antibody,in order to provide reference for formulating the product standard and quality control strategy.Methods:The samples which differ in glycosylation were separated by hydrophobic interaction chromatography(HIC),and the structures were characterized by Circular Dichroism(CD)and Differential Scanning Calorimetry(DSC).Surface plasmon resonance technology(SPR)was then used to determine the binding affinity with TGF-β1.In vitro activity was studied by enzyme-linked immunosorbent assay(ELISA)and reporter gene assay(RGA).Results:The samples with glycosylation differences were obtained,and the glycosylation at N538 site exerted no significant effect to of the bifunctional protein,regarding circular dichroism,thermal stability,affinity,TGF-β1 antigen binding activity and intracellular binding activity of TGF-β1.Conclusion:Glycosylation at N538 site does not affect the quality of targeted PD-L1/TGF-βbispecific antibody products,so the control of glycosylation degree at N538 site can be ignored in the production process.