Maiser Benjamin, Baumgartner Kai, Dismer Florian, Hubbuch Jürgen
Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Oct 1;1002:313-8. doi: 10.1016/j.jchromb.2015.08.027. Epub 2015 Aug 25.
The combination of PEG-protein conjugation and chromatographic separation is generally known as solid-phase or on-column PEGylation and can provide advantages compared to commonly applied batch PEGylation. Even though the concept was already applied by several authors, changes in the isoform distribution compared to liquid-phase PEGylation due to sterically hindered PEGylation sites could not be confirmed. In this manuscript, a method for solid-phase PEGylation experiments in a 96-well plate format, using an automated liquid handling station is described. Applying size exclusion chromatography (SEC) and highly sensitive isoform analytics for mono-PEGylated lysozyme, we were able to investigate the differences in reaction kinetics and isoform distribution between adsorber-based PEGylation and modifications in free solution. Accordingly, solid-phase PEGylation with SP Sepharose FF and XL generally showed a reduced PEGylation reaction. In contrast to the predominant N-terminal PEGylation of lysozyme in liquid phase, a main modification of lys 97 and lys 116 was found for solid-phase experiments, which could be explained by binding orientations on corresponding adsorbent materials. Further experiments with varying amounts of bound protein additionally showed an influence on the isoform distribution of mono-PEGylated lysozyme.
聚乙二醇（PEG）与蛋白质的偶联和色谱分离相结合通常被称为固相或柱上聚乙二醇化，与常用的批量聚乙二醇化相比具有优势。尽管已有多位作者应用了这一概念，但由于空间位阻的聚乙二醇化位点，与液相聚乙二醇化相比，异构体分布的变化尚未得到证实。在本论文中，描述了一种使用自动液体处理工作站在96孔板中进行固相聚乙二醇化实验的方法。通过应用尺寸排阻色谱（SEC）和对单聚乙二醇化溶菌酶进行高灵敏度的异构体分析，我们能够研究基于吸附剂的聚乙二醇化与游离溶液中修饰之间的反应动力学和异构体分布差异。因此，用SP Sepharose FF和XL进行的固相聚乙二醇化通常显示聚乙二醇化反应减少。与液相中溶菌酶主要在N端进行聚乙二醇化不同，固相实验中发现主要修饰位点是lys 97和lys 116，这可以通过相应吸附材料上的结合方向来解释。用不同量的结合蛋白进行的进一步实验还显示了对单聚乙二醇化溶菌酶异构体分布的影响。
