不同细胞系表达的抗EGFR单抗糖基化结构对比分析

发布时间：2018-01-05 20:12

本文关键词：不同细胞系表达的抗EGFR单抗糖基化结构对比分析　出处：《生物工程学报》2017年06期 　论文类型：期刊论文

【摘要】：真核表达系统造就了单克隆抗体药物的广泛异质性,这些异质性通常是由翻译后修饰引起,而糖基化修饰则是关键的翻译后修饰,其对治疗性蛋白的安全性和有效性有着深远的影响,为探索细胞表达系统的改变对单抗糖基化所带来的影响,应用液相色谱-电喷雾离子化四极杆飞行时间质谱技术(LC-ESI-Q-Tof),通过交替高低碰撞能量扫描、源内诱导解离及二级质谱的方法从释放的寡聚糖水平研究聚糖结构,对比分析由两种不同细胞系制备的抗表皮生长因子受体(EGFR)单抗,然后结合外切糖苷酶逐级消化的方法对两种蛋白的糖链结构作进一步确证分析。分析结果表明,在Fc区域的糖基化修饰,两种表达系统表达的该抗体未发生明显的改变,而在Fab区域,由小鼠骨髓瘤细胞SP2/0制备的抗EGFR单抗的聚糖结构中含有大量α半乳糖(α-Gal),且末端唾液酸形式主要是N-羟乙基神经氨酸(NGNA),具有极高的免疫原性风险。而通过中国仓鼠卵巢细胞CHO表达系统制备的抗EGFR单抗Fab区域聚糖结构中不含有α-Gal,且末端唾液酸形式主要是N乙酰神经氨酸(NANA),免疫原性风险极大降低。本研究在一定程度上可以预测由CHO表达系统制备的抗EGFR单抗具备较好的临床耐受性,超敏反应发生风险低,CHO细胞可以作为该抗体改良型生物类似药(Biobetter)的优选表达系统。
[Abstract]:Eukaryotic expression systems have created a wide range of heterogeneity of monoclonal antibody drugs. These heterogeneity is usually caused by post-translational modification, while glycosylation is the key posttranslational modification. It has a profound effect on the safety and efficacy of therapeutic proteins, and it is important to explore the effect of the change of cell expression system on the glycosylation of monoclonal antibodies. Liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (TTOMS) technique was used to scan LC-ESI-Q-TofU by alternating high and low collision energy. In vitro induced dissociation and secondary mass spectrometry were used to study the structure of polysaccharide at the level of released oligosaccharides, and to compare and analyze the EGFR monoclonal antibodies prepared from two different cell lines. The sugar chain structure of the two proteins was further confirmed and analyzed by the method of exoglucosidase step by step digestion. The results showed that the glycosylation modification in FC region. The antibody expressed by the two expression systems did not change significantly, but in the Fab region. A large number of 伪 -galactose (伪 -galactose) were found in the polysaccharide structure of anti-#en1# monoclonal antibody prepared from mouse myeloma cell line SP2/0. The main form of terminal sialic acid is N-hydroxyethyl neuraminic acid (NGNA). There is a high risk of immunogenicity, but 伪 -Gal is not found in the Fab region of anti EGFR monoclonal antibody prepared by CHO expression system of Chinese hamster ovary cells. The main form of terminal sialic acid is N-acetylneuraminic acid (NANAA). The immunogenicity risk is greatly reduced. To some extent, this study can predict that anti-#en1# monoclonal antibody prepared by CHO expression system has better clinical tolerance, and the risk of hypersensitivity reaction is low. CHO cells can be used as a selective expression system for the modified bioanalogues of the antibody.
【作者单位】：上海药品审评核查中心;抗体药物与靶向治疗国家重点实验室;
【基金】：研发公共服务平台(No.16DZ2292900) 产学研医(No.16DZ1910400) 科学仪器领域(No.16142201700) 企业国际合作项目(No.16430730400) 上海市生物医药领域科技支撑项目(Nos.16431904100,16431901200,16431904700) 上海青年科技启明星项目(No.16QB1404300)资助~~
【分类号】：R392
【正文快照】： Received:February 26,2017;Accepted:April 6,2017Supported by:RD Public Service Platform(No.16DZ2292900),Industry School Research Medicine(No.16DZ1910400),ScientificInstrument Field(No.16142201700),Enterprise International Cooperation Projects(No.16430730

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