雷珠单抗的大肠埃希菌胞外分泌表达、纯化及结构表征

Extracellular Expression and Purification of Ranibizumab in Escherichia coli and Its Structural Characterization

抗血管内皮细胞生长因子(VEGF)抗体可抑制新生血管的形成和生长,在年龄相关性黄斑变性眼科疾病的临床治疗中具有重要应用价值,特别是已上市的商品名为雷珠单抗的VEGF抗体Fab片段,在市场上取得巨大成功。本研究探索了雷珠单抗在大肠埃希菌中的胞外分泌表达技术,所表达的产品利用SDS-PAGE、Western Blot、肽谱图全序列分析和纳米差示扫描量热法(Nano DSC)等手段进行了初步的结构表征。结果显示,本研究成功实现了雷珠单抗的大肠埃希菌胞外分泌表达,经过进一步的Capto L亲和色谱获得了有正确氨基酸序列的Fab产品,经质谱分析具有正确的二硫键配对,且具有与标准品相似的热稳定性,为其进一步的工业生产奠定了基础。

Anti-vascular endothelial growth factor(VEGF) antibody can inhibit the angiogenesis, which has great significance in the clinical treatment of ophthalmic diseases such as age-related macular degeneration(AMD). The anti-VEGF Fab drug, ranibizumab, has achieved great success in market. In this study, we explored the extracellular expression technique of ranibizumab. The product was characterized by SDS-PAGE, Western Blot, UPLC-MS peptide mapping, and nano differential scanning calorimetry(Nano DSC). The results demonstrated that the ranibizumab was expressed and secreted into medium efficiently, which was further purified by Capto L affinity chromatography. The obtained product was determined to be with correct amino acid sequence and thermal stability in comparison with the standard reference, which laid a foundation for its further industrial manufacturing.