Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, Vienna, Austria.
Biotechnol J. 2012 May;7(5):635-41. doi: 10.1002/biot.201100407.
PEGylation is the most successful approach, to date, to prolong the in vivo survival of recombinant proteins. The conjugation of the polymer to glycoproteins results in challenging analysis, and furthermore, requires a wide variety of analytical tools for the determination of the extent of PEGylation. Herein, we present microchip capillary gel electrophoresis (MCGE) with a non-commercial high-molecular-weight protein assay for the analysis of the PEGylation degree with a focus on multiple PEGylation. To show the potential of the modified MCGE system, high-mass PEGylated glycoproteins (e.g. coagulation factor VIII) were analyzed. For the von Willebrand factor, the influence of glycans and the hydrodynamic radius on migration time and molecular weight determination is shown. The modified MCGE assay system is a powerful tool for the rapid assessment of the degree of PEGylation, demonstrating conjugate quality or reaction control of PEGylated proteins. This is the main advantage over time-consuming conventional SDS-PAGE. Furthermore, electrophoretic separation, staining, destaining, and fluorescence detection in one step combined with automated data analysis show that the MCGE system is a promising technique for high-throughput monitoring. The MCGE system can be used for rapid structure confirmation ("MCGE fingerprinting") of multiply PEGylated glycoproteins beyond the 230 kDa molecular mass range.
聚乙二醇化是迄今为止延长重组蛋白体内半衰期最成功的方法。聚合物与糖蛋白的连接导致分析具有挑战性，此外，需要广泛的分析工具来确定聚乙二醇化的程度。在此，我们提出了微芯片毛细管凝胶电泳（MCGE）与非商业的高分子量蛋白质分析方法，用于分析聚乙二醇化程度，重点是多聚乙二醇化。为了展示改良的 MCGE 系统的潜力，分析了高分子量聚乙二醇化糖蛋白（如凝血因子 VIII）。对于血管性血友病因子，显示了聚糖和流体力学半径对迁移时间和分子量测定的影响。改良的 MCGE 分析系统是快速评估聚乙二醇化程度的有力工具，可用于评估聚乙二醇化蛋白的缀合物质量或反应控制。这是相对于耗时的传统 SDS-PAGE 的主要优势。此外，电泳分离、染色、脱色和荧光检测一步完成，并结合自动化数据分析，表明 MCGE 系统是高通量监测的有前途的技术。MCGE 系统可用于快速确认（“MCGE 指纹分析”）多聚乙二醇化糖蛋白的结构，超出 230 kDa 的分子量范围。
