Gawlitzek M, Valley U, Wagner R
Cell Culture Technology Department, GBF-Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, D-38124 Braunschweig,
Biotechnol Bioeng. 1998 Mar 5;57(5):518-28.
The effect of different ammonium concentrations and glucosamine on baby hamster kidney (BHK)-21 cell cultures grown in continuously perfused double membrane bioreactors was investigated with respect to the final carbohydrate structures of a secretory recombinant glycoprotein. The human interleukin-2 (IL-2) mutant glycoprotein variant IL-Mu6, which bears a novel N-glycosylation site (created by a single amino acid exchange of Gln100 to Asn), was produced under different defined protein-free culture conditions in the presence or absence of either glutamine, NH4Cl, or glucosamine. Recombinant glycoprotein products were purified and characterized by amino acid sequencing and carbohydrate structural analysis using matrix-assisted laser desorption ionization time of flight mass spectrometry, high-pH anion-exchange chromatography with pulsed amperometric detection, and methylation analysis. In the absence of glutamine, cells secreted glycoprotein forms with preponderantly biantennary, proximal fucosylated carbohydrate chains (85%) with a higher NeuAc content (58%). Under standard conditions in the presence of 7.5 mM glutamine, complex-type N-glycans were found to be mainly biantennary (68%) and triantennary structures (33%) with about 50% containing proximal alpha1-6-linked fucose; 37% of the antenna were found to be substituted with terminal alpha2-3-linked N-acetylneuraminic acid. In the presence of 15 mM exogenously added NH4Cl, a significant and reproducible increase in tri- and tetraantennary oligosaccharides (45% of total) was detected in the secretion product. In glutamin-free cultures supplemented with glucosamine, an intermediate amount of high antennary glycans was detected. The increase in complexity of N-linked oligosaccharides is considered to be brought about by the increased levels of intracellular uridine diphosphate-GlcNAc/GalNAc. These nucleotide sugar pools were found to be significantly elevated in the presence of high NH3/NH4+ and glucosamine concentrations.
针对一种分泌型重组糖蛋白的最终碳水化合物结构，研究了不同铵浓度和氨基葡萄糖对在连续灌注双膜生物反应器中培养的幼仓鼠肾（BHK）-21细胞培养物的影响。人白细胞介素-2（IL-2）突变糖蛋白变体IL-Mu6带有一个新的N-糖基化位点（由Gln100单氨基酸交换为Asn产生），在不同定义的无蛋白培养条件下，在有或没有谷氨酰胺、NH4Cl或氨基葡萄糖的情况下产生。重组糖蛋白产物通过氨基酸测序以及使用基质辅助激光解吸电离飞行时间质谱、带脉冲安培检测的高pH阴离子交换色谱和甲基化分析进行碳水化合物结构分析来进行纯化和表征。在没有谷氨酰胺的情况下，细胞分泌的糖蛋白形式主要是带有较高NeuAc含量（58%）的双天线、近端岩藻糖基化碳水化合物链（85%）。在存在7.5 mM谷氨酰胺的标准条件下，发现复合型N-聚糖主要是双天线结构（68%）和三天线结构（33%），约50%含有近端α1-6连接的岩藻糖；37%的天线被发现被末端α2-3连接的N-乙酰神经氨酸取代。在添加15 mM外源NH4Cl的情况下，在分泌产物中检测到三天线和四天线寡糖显著且可重复增加（占总量的45%）。在补充有氨基葡萄糖的无谷氨酰胺培养物中，检测到中等数量的高天线聚糖。N-连接寡糖复杂性的增加被认为是由细胞内尿苷二磷酸-GlcNAc/GalNAc水平的增加引起的。发现在高NH3/NH4+和氨基葡萄糖浓度存在的情况下，这些核苷酸糖库显著升高。
