Department of Analytical Chemistry, Stockholm University, 106 91 Stockholm, Sweden.
Anal Bioanal Chem. 2011 Feb;399(4):1601-11. doi: 10.1007/s00216-010-4469-y. Epub 2010 Dec 21.
Recombinant therapeutic antibodies have shown a great potential in the treatment of several severe medical conditions such as cancer and autoimmune diseases. Glycosylation plays a critical role in biological activity and immunogenic properties of these compounds. The analysis of glycan profiles is therefore necessary in many steps of the development and manufacturing process from early development to quality control of the final product. In this paper, a fast, parallel, and robust sample preparation platform for glycosylation profiling using a microfluidic compact disc (CD) is presented. A sequential process including selective capture of antibody from a crude cell supernatant using protein A beads, enzymatic release of glycans, purification with a graphitized carbon black column, and crystallisation for MALDI-TOF analysis were performed on the CD. Glycosylation profiles of an antibody intended for therapeutic use produced in two different cell lines were compared.
重组治疗性抗体在治疗癌症和自身免疫性疾病等多种严重疾病方面显示出巨大的潜力。糖基化在这些化合物的生物活性和免疫原性方面起着关键作用。因此，在从早期开发到最终产品的质量控制的开发和制造过程的许多步骤中，都需要分析聚糖图谱。本文提出了一种使用微流控光盘（CD）进行糖基化分析的快速、平行和稳健的样品制备平台。该方法包括使用蛋白 A 珠从粗细胞上清液中选择性捕获抗体、用酶释放聚糖、用石墨化碳黑柱纯化以及用于 MALDI-TOF 分析的结晶等顺序过程。比较了在两种不同细胞系中生产的一种治疗用抗体的糖基化图谱。
