Wang Chong, Guo Huaizu
Shanghai Center for Drug Evaluation and Inspection, Shanghai 201203, China.
State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai 201203, China.
Sheng Wu Gong Cheng Xue Bao. 2017 Jun 25;33(6):1018-1027. doi: 10.13345/j.cjb.170074.
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.
哺乳动物表达系统的使用导致单克隆抗体（mAb）产品存在显著的异质性，这通常归因于翻译后修饰，而糖基化是一种关键的翻译后修饰，因为它对mAb的安全性和有效性有深远影响。本研究旨在探讨不同表达系统对mAb N-糖基化的影响。使用液相色谱电喷雾电离四极杆飞行时间质谱（LC-ESI-QTof MS）在游离寡糖水平上成功表征了不同表达系统产生的抗表皮生长因子受体（EGFR）单克隆抗体之间各个聚糖的详细结构。还采用了交替的低碰撞能量和高碰撞能量扫描、源内碰撞诱导解离以及与外切糖苷酶消化法相结合的串联质谱（MS/MS）。综合数据显示，CHO细胞表达系统产生的抗EGFR抗体的Fab区域糖基化模式与SP2/0细胞表达系统不同，而Fc区域基本保持不变。我们证实，SP2/0细胞表达系统产生的抗EGFR抗体具有更多样化的带有α-半乳糖的聚糖混合物以及一种不期望的、异常形式的唾液酸化N-羟乙酰神经氨酸（NGNA）。在主要含有唾液酸N-乙酰神经氨酸（NANA）的CHO细胞表达系统产生的mAb中不存在α-半乳糖，NANA是期望的、正常的人源型唾液酸化。本研究从理论上预测，CHO细胞表达系统产生的抗EGFR抗体可能表现出更好的临床耐受性以及极低的主动超敏反应可能性，CHO细胞系可以成为生产抗EGFR生物类似药的首选表达系统。
