A cost-effective plate-based sample preparation for antibody N-glycan analysis
A cost-effective plate-based sample preparation for antibody N-glycan analysis
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DOI：
10.1016/j.chroma.2013.07.104
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发表时间：
2013-09-13
影响因子：
4.1
通讯作者：
Gong, Bing
中科院分区：
文献类型：
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作者：
Burnina, Irina;Hoyt, Erik;Gong, Bing
在治疗性抗体的早期细胞系和工艺开发期间，考虑到需要分析大量样品，非常需要一种具有成本效益的高通量方法来表征N-连接聚糖。使用市售的低成本96孔板，我们开发了一种实用的程序来制备荧光标记的N-连接聚糖，用于通过质谱（MS）和高效液相色谱（UPLC）进行定性和定量分析。抗体样品在单个96孔疏水膜滤板中连续变性、还原和去糖基化。随后，将释放的聚糖在收集板中进行荧光标记，并使用亲水性膜滤板进行清除。完全在具有简单缓冲系统的即用型96孔板中进行，该过程需要不到90分钟即可完成。我们应用优化的程序来检查曲妥珠单抗的N-连接糖基化，并能够定量10个主要的N-连接聚糖。不同量起始物料（10-200 μ g）的结果高度相似，表明该方法具有耐用性。与其他方法相比，这种新方法易于实施，经济上更实惠，并且对于抗体开发的早期筛查非常有价值。(C)2013爱思唯尔有限公司版权所有。
During early cell line and process development of therapeutic antibodies, a cost-effective high-throughput approach to characterize the N-linked glycans is highly desired given that a large number of samples need to be analyzed. Using commercially available, low cost 96-well plates, we developed a practical procedure to prepare fluorescently labeled N-linked glycans for both qualitative and quantitative analysis by mass spectrometry (MS) and ultrahigh performance liquid chromatography (UPLC). Antibody samples were continuously denatured, reduced, and deglycosylated in a single 96-well hydrophobic membrane filter plate. Subsequently, released glycans were fluorescently labeled in a collection plate, and cleaned-up using a hydrophilic membrane filter plate. Carried out entirely in ready-to-use 96-well plates with simple buffer systems, this procedure requires less than 90 min to finish. We applied the optimized procedure to examine the N-linked glycosylation of trastuzumab and were able to quantify ten major N-linked glycans. The results from different amounts of starting materials (10-200 mu g) were highly similar and showed the robustness of this procedure. Compared to other methods, this new procedure is simple to implement, economically more affordable, and could be very valuable for early screenings of antibody development. (C) 2013 Elsevier B.V. All rights reserved.