Aghamohseni Hengameh, Ohadi Kaveh, Spearman Maureen, Krahn Natalie, Moo-Young Murray, Scharer Jeno M, Butler Mike, Budman Hector M
Chemical Engineering Department, University of Waterloo, Waterloo, ON, Canada.
Microbiology Department, University of Manitoba, Winnipeg, MB, Canada.
J Biotechnol. 2014 Sep 30;186:98-109. doi: 10.1016/j.jbiotec.2014.05.024. Epub 2014 Jul 8.
The impact of operating conditions on the glycosylation pattern of humanized camelid monoclonal antibody, EG2-hFc produced by Chinese hamster ovary (CHO) cells has been evaluated by a combination of experiments and modeling. Cells were cultivated under different levels of glucose and glutamine concentrations with the goal of investigating the effect of nutrient depletion levels and ammonia build up on the cell growth and the glycoprofiles of the monoclonal antibody (Mab). The effect of average pH reduction on glycosylation level during the entire culture time or during a specific time span was also investigated. The relative abundance of glycan structures was quantified by hydrophilic interaction liquid chromatography (HILIC) and the galactosylation index (GI) and the sialylation index (SI) were determined. Lower initial concentrations of glutamine resulted in lower glucose consumption and lower cell yield but increased GI and SI levels when compared to cultures started with higher initial glutamine levels. Similarly, reducing the average pH of culture resulted in lower growth but higher SI and GI levels. These findings indicate that there is a tradeoff between cell growth, resulting Mab productivity and the achievement of desirable higher glycosylation levels. A dynamic model, based on a metabolic flux analysis (MFA), is proposed to describe the metabolism of nutrients, cell growth and Mab productivity. Finally, existing software (GLYCOVIS) that describes the glycosylation pathways was used to illustrate the impact of extracellular species on the glycoprofiles.
通过实验与建模相结合的方式，评估了操作条件对中国仓鼠卵巢（CHO）细胞产生的人源化骆驼科单克隆抗体EG2-hFc糖基化模式的影响。在不同葡萄糖和谷氨酰胺浓度水平下培养细胞，目的是研究营养物质消耗水平和氨积累对细胞生长以及单克隆抗体（Mab）糖谱的影响。还研究了在整个培养时间或特定时间段内平均pH降低对糖基化水平的影响。通过亲水相互作用液相色谱（HILIC）对聚糖结构的相对丰度进行定量，并测定半乳糖基化指数（GI）和唾液酸化指数（SI）。与初始谷氨酰胺水平较高的培养物相比，较低的初始谷氨酰胺浓度导致较低的葡萄糖消耗和较低的细胞产量，但提高了GI和SI水平。同样，降低培养的平均pH导致生长较慢，但SI和GI水平较高。这些发现表明，在细胞生长、所得Mab生产力与实现理想的更高糖基化水平之间存在权衡。提出了一个基于代谢通量分析（MFA）的动态模型来描述营养物质的代谢、细胞生长和Mab生产力。最后，使用描述糖基化途径的现有软件（GLYCOVIS）来说明细胞外物质对糖谱的影响。
