Institute of Technology and Science, The University of Tokushima, Minamijosanjima-cho 2-1, Tokushima 770-8506, Japan.
Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka 2-1, Suita, Osaka 565-0871, Japan.
J Biosci Bioeng. 2014 May;117(5):639-44. doi: 10.1016/j.jbiosc.2013.11.001. Epub 2013 Dec 8.
N-Glycosylation of therapeutic antibodies contributes not only to their biological function, but also to their stability and tendency to aggregate. Here, we investigated the impact of the glycosylation status of an aggregated antibody that accumulated during the bioreactor culture of Chinese hamster ovary cells. High-performance liquid chromatography analysis showed that there was no apparent difference in the glycosylation patterns of monomeric, dimeric, and large aggregated forms of the antibody. In contrast, lectin binding assays, which enable the total amounts of specific sugar residues to be detected, showed that both galactose and fucose residues in dimers and large aggregates were reduced to 70-80% of the amount in monomers. These results strongly suggest that the lack of N-linked oligosaccharides, a result of deglycosylation or aglycosylation, occurred in a proportion of the dimeric and large aggregated components. The present study demonstrates that glycosylation heterogeneities are a potential cause of antibody aggregation in cell culture of Chinese hamster ovary cells, and that the lack of N-glycosylation promotes the formation of dimers and finally results in large aggregates.
N-糖基化不仅影响治疗性抗体的生物学功能，也影响其稳定性和聚集倾向。在这里，我们研究了在仓鼠卵巢细胞生物反应器培养过程中积累的聚集抗体的糖基化状态的影响。高效液相色谱分析表明，抗体的单体、二聚体和大聚集形式之间的糖基化模式没有明显差异。相比之下，凝集素结合测定法可以检测到特定糖残基的总量，结果表明二聚体和大聚集体中的半乳糖和岩藻糖残基减少到单体中糖残基数量的 70-80%。这些结果强烈表明，在二聚体和大聚集成分中，存在 N 连接寡糖的缺失，这是脱糖基化或无糖基化的结果。本研究表明，糖基化异质性是仓鼠卵巢细胞培养中抗体聚集的一个潜在原因，并且 N-糖基化的缺失促进了二聚体的形成，最终导致了大聚集体的形成。
