Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, China.
College of Basic Medical Sciences, Dalian Medical University, Dalian, Liaoning 116044, China.
J Biochem. 2021 Sep 22;170(1):139-151. doi: 10.1093/jb/mvab051.
Glycoprotein therapeutics are among the leading products in the biopharmaceutical industry. The heterogeneity of glycans in therapeutic proteins is an issue for maintaining quality, activity and safety during bioprocessing. In this study, we knocked out genes encoding Golgi α-mannosidase-II, MAN2A1 and MAN2A2 in human embryonic kidney 293 (HEK293) cells, establishing an M2D-KO cell line that can produce recombinant proteins mainly with hybrid-type N-glycans. Furthermore, FUT8, which encodes α1,6-fucosyltransferase, was knocked out in the M2D-KO cell line, establishing a DF-KO cell line that can express noncore fucosylated hybrid-type N-glycans. Two recombinant proteins, lysosomal acid lipase and constant fragment of human IgG1, were expressed in the M2D-KO and DF-KO cell lines. Glycan structural analysis revealed that complex-type N-glycans were removed in both M2D-KO and DF-KO cells. Our results suggest that these cell lines are suitable for the production of therapeutic proteins with hybrid-type N-glycans. Moreover, KO cell lines would be useful as models for researching the mechanism of antimetastatic effects in human tumours by swainsonine treatment.
糖蛋白治疗药物是生物制药行业的主要产品之一。治疗蛋白中聚糖的异质性是在生物工艺过程中保持质量、活性和安全性的一个问题。在这项研究中，我们敲除了人胚肾 293（HEK293）细胞中编码高尔基α-甘露糖苷酶-II、MAN2A1 和 MAN2A2 的基因，建立了一个能够主要产生具有杂合型 N-聚糖的重组蛋白的 M2D-KO 细胞系。此外，我们还敲除了 M2D-KO 细胞系中的编码α1,6-岩藻糖基转移酶的 FUT8 基因，建立了一个能够表达非核心岩藻糖化杂合型 N-聚糖的 DF-KO 细胞系。两种重组蛋白，溶酶体酸性脂肪酶和人 IgG1 的恒定片段，在 M2D-KO 和 DF-KO 细胞系中表达。糖基结构分析表明，复杂型 N-聚糖在 M2D-KO 和 DF-KO 细胞中均被去除。我们的结果表明，这些细胞系适合生产具有杂合型 N-聚糖的治疗性蛋白。此外，KO 细胞系可用作研究长春瑞滨治疗人类肿瘤转移抑制作用机制的模型。
