Baker K N, Rendall M H, Hills A E, Hoare M, Freedman R B, James D C
Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK.
Biotechnol Bioeng. 2001 May 5;73(3):188-202. doi: 10.1002/bit.1051.
Chinese hamster ovary and murine myeloma NS0 cells are currently favored host cell types for the production of therapeutic recombinant proteins. In this study, we compared N-glycan processing in GS-NS0 and GS-CHO cells producing the same model recombinant glycoprotein, tissue inhibitor of metalloproteinases 1. By manipulation of intracellular nucleotide-sugar content, we examined the feasibility of implementing metabolic control strategies aimed at reducing the occurrence of murine-specific glycan motifs on NS0-derived recombinant proteins, such as Galalpha1,3Galbeta1,4GlcNAc. Although both CHO and NS0-derived oligosaccharides were predominantly of the standard complex type with variable sialylation, 30% of N-glycan antennae associated with NS0-derived TIMP-1 terminated in alpha1,3-linked galactose residues. Furthermore, NS0 cells conferred a greater proportion of terminal N-glycolylneuraminic (sialic) acid residues as compared with the N-acetylneuraminic acid variant. Inclusion of the nucleotide-sugar precursors, glucosamine (10 mM, plus 2 mM uridine) and N-acetylmannosamine (20 mM), in culture media were shown to significantly increase the intracellular pools of UDP-N-acetylhexosamine and CMP-sialic acid, respectively, in both NS0 and CHO cells. The elevated UDP-N-acetylhexosamine content induced by the glucosamine/uridine treatment was associated with an increase in the antennarity of N-glycans associated with TIMP-1 produced in CHO cells but not N-glycans associated with TIMP-1 from NS0 cells. In addition, elevated UDP-N-acetylhexosamine content was associated with a slight decrease in sialylation in both cell lines. The elevated CMP-sialic acid content induced by N-acetylmannosamine had no effect on the overall level of sialylation of TIMP-1 produced by both CHO and NS0 cells, although the ratio of N-glycolylneuraminic acid:N-acetylneuraminic acid associated with NS0-derived TIMP-1 changed from 1:1 to 1:2. These data suggest that manipulation of nucleotide-sugar metabolism can promote changes in N-glycan processing that are either conserved between NS0 and CHO cells or specific to either NS0 cells or CHO cells.
中国仓鼠卵巢细胞和鼠骨髓瘤NS0细胞是目前用于生产治疗性重组蛋白的首选宿主细胞类型。在本研究中，我们比较了产生相同模型重组糖蛋白金属蛋白酶组织抑制剂1的GS-NS0细胞和GS-CHO细胞中的N-聚糖加工过程。通过操纵细胞内核苷酸糖含量，我们研究了实施代谢控制策略以减少NS0来源的重组蛋白上鼠特异性聚糖基序（如Galα1,3Galβ1,4GlcNAc）出现的可行性。尽管CHO和NS0来源的寡糖主要是具有可变唾液酸化的标准复合型，但与NS0来源的TIMP-1相关的N-聚糖触角中有30%以α1,3-连接的半乳糖残基终止。此外，与N-乙酰神经氨酸变体相比，NS0细胞赋予的末端N-羟乙酰神经氨酸（唾液酸）残基比例更高。在培养基中加入核苷酸糖前体氨基葡萄糖（10 mM，加2 mM尿苷）和N-乙酰甘露糖胺（20 mM），结果显示在NS0细胞和CHO细胞中分别显著增加了UDP-N-乙酰己糖胺和CMP-唾液酸的细胞内池。氨基葡萄糖/尿苷处理诱导的UDP-N-乙酰己糖胺含量升高与CHO细胞中产生的TIMP-1相关的N-聚糖触角增加有关，但与NS0细胞中TIMP-1相关的N-聚糖无关。此外，UDP-N-乙酰己糖胺含量升高与两种细胞系中唾液酸化的轻微降低有关。N-乙酰甘露糖胺诱导的CMP-唾液酸含量升高对CHO和NS0细胞产生的TIMP-1的总体唾液酸化水平没有影响，尽管与NS0来源的TIMP-1相关的N-羟乙酰神经氨酸：N-乙酰神经氨酸的比例从1:1变为1:2。这些数据表明，操纵核苷酸糖代谢可以促进NS0细胞和CHO细胞之间保守的或NS0细胞或CHO细胞特有的N-聚糖加工变化。
