用表面等离子体共振技术评价糖基化在人IgG4抗体中的功能作用

R. B. Wong, M. Zeng, an-Horng Lee, T. S. Raju, K. Cheng
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引用次数: 1

摘要

免疫球蛋白的Fc糖基化是抗体效应作用所必需的。这些免疫球蛋白的聚糖通常被称为糖型,由于存在于内质网(ER)和高尔基体中的糖基化机制的变化,它们可能是异质的。在缺乏一个通用的培养方案,可以呈现一致的糖基化模式,监测单克隆抗体的糖型从培养细胞是必不可少的。因此,使用Agilent生物分析仪lab - on - chip电泳系统完成IgG4单克隆抗体糖基化和去糖基化重链的定量。除天然抗体外,用PNGase F和F(ab')2片段处理制备的完全去糖基化抗体,用Biacore表面等离子体共振(SPR)评价其抗原结合动力学。结果表明,天然抗体和去糖基化抗体的平衡结合常数KD分别为1.81E-09M和1.96E-09M, F(ab’)2的平衡结合常数KD为5.79E-10M。采用竞争结合测定法的体外生物活性也被开发来证明Fc聚糖的作用。结果证实,对于治疗性中和抗体,天然单克隆抗体-1和去糖基化抗体的生物活性是相当的,从而表明Fc聚糖不参与抗原结合或生物学功能。在SPR仪器上进行的动力学和竞争性分析快速可靠。与片上电泳法相结合,可作为工艺开发和质量控制的监测方法。
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Functional Role of Glycosylation in a Human IgG4 Antibody Assessed by Surface Plasmon Resonance Technology
Fc glycosylation of immunoglobulins is necessary for antibody effector functions. These glycans of immunoglobulins are often referred as glycoforms and they can be heterogeneous due to the variations in glycosylation machinery present in the endoplastic reticulum (ER) and in the Golgi. In the absence of a generic culturing protocol that can render a consistent glycosylation pattern, monitoring glycoforms of monoclonal antibodies from cultured cells is becoming essential. Accordingly, quantification of glycosylated and deglycosylated heavy chains of an IgG4 monoclonal antibody was accomplished using an Agilent Bioanalyzer Lab-On-the-Chip electrophoresis system. In addition to the native antibody, completely deglycosylated antibody prepared by treating with PNGase F and a F(ab')2 fraction were evaluated for their antigen binding kinetics using Biacore surface Plasmon resonance (SPR). The equilibrium binding constants KD are found to be comparable at 1.81E-09M, 1.96E-09M, for the native and deglycosylated antibody, respectively, and 5.79E-10M for the F(ab')2. An in vitro biological activity employing a competition binding assay was also developed to demonstrate the role of the Fc glycan. The results confirm that for a neutralizing antibody therapeutic the biological activity of the native MAb-1 and deglycosylated antibody are comparable, thus indicating that the Fc glycan does not contribute to the antigen binding or the biological function. The kinetics and competitive assays performed on an SPR instrument are quick and reliable. Combined with the on-chip electrophoresis method they can be used as monitoring methods for process development and quality control.
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