氨浓度升高对CHO细胞维持期抗体融合蛋白的表达及N-糖基化的影响

Effects of Elevated Ammonia Concentration on Production and N-glycosylation of Antibody Fusion Protein in CHO Cell Cultures at Maintenance Phase

为了深入理解在中国仓鼠卵巢(CHO)细胞流加培养过程中氨对抗体融合蛋白表达和N-糖基化的作用,认识氨影响N-糖基化加工的作用位点,考察了在细胞维持期(产物表达期)不同氨浓度条件与CHO细胞维持与代谢、抗体融合蛋白表达和N-糖链结构的关系。结果显示,氨浓度在5-12 mmol/L范围内对维持期的细胞生长曲线、葡萄糖和谷氨酰胺的消耗以及乳酸和氨的生成情况没有明显影响。但当氨浓度大于5 mmol/L时,随着氨浓度的升高,抗体融合蛋白的唾液酸化程度和半乳糖化程度均不断降低,而岩藻糖基化程度和高甘露糖糖型比例则没有变化。当氨浓度升高至大于9 mmol/L后,抗体融合蛋白的表达能力和最终表达量开始降低。因此,应在细胞培养工艺过程开发时控制氨的生成至小于5 mmol/L,以避免氨的累积导致产物的半乳糖化和唾液酸化程度降低以及产物表达量下降。

In order to understand the effects of ammonia on production and N-glycosylation of antibody fusion protein(AFP)during fedbatch cultures of Chinese hamster ovary(CHO)cells and to reveal the N-glycosylation steps affected by ammonia,the CHO cell maintenance and metabolism,protein production,and N-glycan structure of AFP at different ammonia concentrations were investigated during cell maintenance phase(protein production phase).It was found that cell growth profile,glucose and glutamine consumption,production level of lactate and ammonia during cell maintenance phase were insignificantly affected at 5-12 mmol/L of ammonia concentration.However,elevated ammonia concentration(>5 mmol/L)led to decreased sialylation and galactosylation but no effect on fucosylation and high mannose species of AFP.The productivity and final titer of AFP decreased while ammonia concentration was>9 mmol/L.Thus,ammonia production should be carefully controlled<5 mmol/L during the development of cell culture process to avoid impaired galactosylation and sialylation of the product and decreased product expression from ammonia accumulation.