Capillary electrophoresis with laser-induced fluorescence detection for detailed studies on N-linked oligosaccharide profile of therapeutic recombinant monoclonal antibodies
Capillary electrophoresis with laser-induced fluorescence detection for detailed studies on N-linked oligosaccharide profile of therapeutic recombinant monoclonal antibodies
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DOI：
10.1016/j.chroma.2006.08.028
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发表时间：
2006-11-10
影响因子：
4.1
通讯作者：
Kakehi, Kazuaki
中科院分区：
文献类型：
--
作者：
Kamoda, Satoru;Ishikawa, Rika;Kakehi, Kazuaki
建立了重组单克隆抗体（rmAb）总N-连接寡糖的毛细管电泳-激光诱导荧光检测（CE-LIF）分析方法和N-连接寡糖的结构分析方法。使用2-氨基苯甲酸（2-AA）作为荧光试剂的CE-LIF方法，在分析来自市售rmAb药物利妥昔单抗的N-连接寡糖时，除了典型的无唾液酸-双触角复合型寡糖外，还可以灵敏地检测几个次要峰。通过与分别通过高效液相色谱（HPLC）和基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）分离和测定的2-AA衍生化寡糖的迁移时间进行比较，这些次要峰被成功地指定为唾液酸-双触角复合物型和高甘露糖型寡糖。在生物药物开发中，评价这些次要寡糖非常重要，因为其中一些次要聚糖可能影响体内免疫原性和清除率。使用CE-LIF的重复性分析显示相对校正峰面积具有良好的精密度。这些结果表明，本CE-LIF方法适用于来自rmAb药物的N-连接寡糖的结构表征和定量分析。本方法将是在rmAb药物开发的各个阶段（如克隆选择、生物工艺控制和常规批放行检测）快速、定量和详尽评价N-连接寡糖的有力工具，以确保产品有效性和一致性。(c)2006 Elsevier B. V.保留所有权利。
Total N-linked oligosaccharide profiling method for recombinant monoclonal antibody (rmAb) using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and an approach for detailed structural analysis of N-linked oligosaccharide were developed. A CE-LIF method using 2-ammobenzoic acid (2-AA) as a fluorogenic reagent allowed sensitive detection of several minor peaks besides typical asialo-biantennary complex type oligosaccharides in the analysis of N-linked oligosaccharide from a commercial rmAb pharmaceutical, rituximab. These minor peaks were successfully assigned as sialo-biantennary complex type and high-mannose type oligosaccharides by comparison with the migration times of 2-AA derivatized oligosaccharides which were separately fractionated and determined by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In development of biopharmaceuticals, it is important to evaluate these minor oligosaccharides, because some of these minor glycans are likely to influence immunogenicity and clearance rate in vivo. The repetitive analysis using CE-LIF showed excellent precision in relative corrected peak areas. These results demonstrate that the present CE-LIF method is applicable for both structural characterization and quantitative profiling of N-linked oligosaccharides derived from rmAb pharmaceuticals. The present method will be a powerful tool for rapid, quantitative and exhaustive evaluation of N-linked oligosaccharides in various stages of rmAb pharmaceutical development such as clone selection, bioprocess control, and routine lot release testing to ensure product efficacy and consistency. (c) 2006 Elsevier B.V. All rights reserved.