不同细胞系表达的抗EGFR单抗糖基化结构对比分析

Characterization of N-glycosylation in an anti-EGFR monoclonal antibody produced by different expression systems

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。

The use of mammalian expression systems results in a remarkable heterogeneity of mAb products, generally due to post-translational modifications, and glycosylation is a critical post-translation modification because it has a profound impact on the safety and efficacy of mAbs. The present study was designed to explore the impact of a different expression system on mAb N-glycosylation. The detailed structures of individual glycans between anti-EGFR monoclonal antibodies produced by different expression systems were successfully characterized at the level of free oligosaccharides using liquid chromatography electrospray ionization quadrupole time-of-fight mass spectrometry （LC-ESI-QTof MS）. An alternating low and elevated collision energy scan, in source collision-induced dissociation and MS/MS in combination with exoglycosidase digestion method was also adopted. The combined data revealed that the Fab region of anti-EGFR antibody produced by CHO cell expression system had a pattern of glycosylation differing from that of the SP2/0 cell expression system whereas the Fc region remained basically unchanged. We confirmed that anti-EGFR antibody produced by SP2/0 cell expression system had a much more diverse mixture of glycans with α-Gal and an undesired, aberrant form of sialylation N-glycolylneuraminic acid （NGNA）. The α-Gal was absent in mAb produced by CHO cell expression system containing sialic acid predominantly N-acetyl neuraminic acid （NANA） which is the desired, normal human-type sialylation. This study theoretically predicts that anti-EGFR antibody produced by CHO cell expression system may show better clinical tolerance, and very low potential for active hypersensitivity reactions, CHO cell lines can be the preferred expression system for producing anti-EGFR biobetter.