Quality control of monoclonal antibodies is challenging due in part to the diversity of post‐translational modifications present. The regulation of the N‐glycans of IgG‐Fc domain is one of the key factors to maintain the safety and efficacy of antibody drugs. The FcγRIIIa affinity column is an attractive tool for the precise analysis of the N‐glycans in IgG‐Fc domain. We used the mutant FcγRIIIa, which is produced in Escherichia coli and is therefore not glycosylated, as an affinity reagent to analyze the N‐glycans of monoclonal antibodies expressed in Expi293 and ExpiCHO cells. The monoclonal antibodies expressed in these cells showed very different chromatograms, because of differences in terminal galactose residues on the IgG‐Fc domains. We also carried out kinetic and thermodynamic analyses to understand the interaction between monoclonal antibodies and the mutant FcγRIIIa. Expi293 cell‐derived monoclonal antibodies had higher affinity for the mutant FcγRIIIa than those derived from ExpiCHO cells, due to slower off rates and lower binding entropy loss. Collectively, our results suggest that the FcγRIIIa column can be used to analyze the glycosylation of antibodies rapidly and specifically.

中文翻译：

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使用 FcγRIIIa 配体对 CHO 和 293 细胞进行 IgG-Fc 结构域 N-聚糖的高灵敏度 HPLC 分析和生物物理表征。

单克隆抗体的质量控制具有挑战性，部分原因在于存在的翻译后修饰的多样性。IgG-Fc结构域N-聚糖的调控是维持抗体药物安全性和有效性的关键因素之一。FcγRIIIa 亲和柱是用于精确分析 IgG-Fc 域中的 N-聚糖的有吸引力的工具。我们使用了在大肠杆菌中产生的突变 FcγRIIIa因此未糖基化，可作为分析 Expi293 和 ExpiCHO 细胞中表达的单克隆抗体的 N-聚糖的亲和试剂。由于 IgG-Fc 结构域上末端半乳糖残基的差异，在这些细胞中表达的单克隆抗体显示出非常不同的色谱图。我们还进行了动力学和热力学分析，以了解单克隆抗体与突变型 FcγRIIIa 之间的相互作用。由于较慢的解离速率和较低的结合熵损失，Expi293 细胞来源的单克隆抗体对突变 FcγRIIIa 的亲和力高于 ExpiCHO 细胞来源的抗体。总的来说，我们的结果表明 FcγRIIIa 色谱柱可用于快速、特异性地分析抗体的糖基化。