Liu Bo, Spearman Maureen, Doering John, Lattová Erica, Perreault Hélène, Butler Michael
Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2.
Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2.
J Biotechnol. 2014 Jan 20;170:17-27. doi: 10.1016/j.jbiotec.2013.11.007. Epub 2013 Nov 25.
The glycosylation pattern of a chimeric heavy chain antibody (EG2) produced from CHO cells was affected by the glucose concentration (0-25mM) of cultures established at high density (>10(6)ml(-1)) over 24h. The resulting proportion of non-glycosylated Mab was directly correlated to the exposure time of cells to media depleted of glucose. Deprivation of glucose for the full 24h resulted in a 45% non-glycosylated Mab fraction. Analysis of steady state levels of intracellular lipid-linked oligosaccharides (LLOs) showed that under glucose limitation there was a reduction in the amount of full length LLO (Glc3Man9GlcNac2), with a concomitant increase in the smaller mannosyl-glycans (Man2-5GlcNAc2). Glycan microheterogeneity was quantified by galactosylation and sialylation indices (GI and SI) which showed a direct correlation to the cell specific glucose uptake. The GI increased to 0.83 following media supplementation with a cocktail of uridine, manganese and galactose. This is significantly higher than for a fully humanized antibody (DP12) produced under the similar conditions or for similar antibodies reported in the literature. The high GI of the chimeric antibody (EG2) may be due to its low molecular weight and unusual structure. These findings are important in relation to the low substrate that may occur in fed-batch cultures for Mab production.
由CHO细胞产生的嵌合重链抗体（EG2）的糖基化模式受到在高密度（>10⁶ml⁻¹）下培养24小时的葡萄糖浓度（0-25mM）的影响。所产生的非糖基化单克隆抗体的比例与细胞暴露于缺乏葡萄糖的培养基中的时间直接相关。在整个24小时内剥夺葡萄糖会导致45%的非糖基化单克隆抗体比例。对细胞内脂质连接寡糖（LLO）稳态水平的分析表明，在葡萄糖限制条件下，全长LLO（Glc3Man9GlcNac2）的量减少，同时较小的甘露糖基聚糖（Man2-5GlcNAc2）增加。通过半乳糖基化和唾液酸化指数（GI和SI）对聚糖微异质性进行定量，结果表明其与细胞特异性葡萄糖摄取直接相关。在用尿苷、锰和半乳糖混合物补充培养基后，GI增加到0.83。这显著高于在类似条件下产生的完全人源化抗体（DP12）或文献中报道的类似抗体。嵌合抗体（EG2）的高GI可能归因于其低分子量和不寻常的结构。这些发现对于在单克隆抗体生产的补料分批培养中可能出现的低底物情况具有重要意义。
