Filep Csenge, Guttman András
Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, Doctoral School of Molecular Medicine, University of Debrecen, 98 Nagyerdei krt, Debrecen H-4032, Hungary.
Translational Glycomics Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, 10 Egyetem u, Veszprem H-8200, Hungary.
Anal Chem. 2021 Feb 23;93(7):3535-3541. doi: 10.1021/acs.analchem.0c04927. Epub 2021 Feb 5.
With the increasing interest in the biopharmaceutical industry toward novel and innovative protein therapeutics, improved separation techniques are important, especially for the analysis of highly glycosylated candidates. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using borate cross-linked dextran is one of the most frequently used methods to analyze biotherapeutic proteins in process control as well as in release and stability testing. In this work, the effect of the monomer (dextran) and cross-linker (borate) ratio was studied in SDS-CGE analysis of a therapeutic monoclonal antibody test item in its reduced and intact forms. A retention model was developed for better understanding of the separation selectivity between the non-glycosylated and glycosylated heavy chain fragments, exploiting the interaction between the dextran-borate adducts and the glycan moiety of the therapeutic antibody. The monomer cross-linker ratio played a significant role in the overall analysis times and affected the separation selectivity between the non-glycosylated and regular (glycosylated) heavy chain fragments; however, it had no effect on the separation of the regular and non-glycosylated intact forms of the monoclonal antibody. Introduction of three-dimensional selectivity plots offered an easy separation optimization option for the separation problem in hand.
随着生物制药行业对新型和创新蛋白质疗法的兴趣日益浓厚，改进的分离技术变得至关重要，特别是对于高度糖基化候选药物的分析。使用硼酸盐交联葡聚糖的十二烷基硫酸钠毛细管凝胶电泳（SDS-CGE）是过程控制以及放行和稳定性测试中分析生物治疗性蛋白质最常用的方法之一。在这项工作中，研究了单体（葡聚糖）与交联剂（硼酸盐）比例对治疗性单克隆抗体测试项目还原形式和完整形式的SDS-CGE分析的影响。开发了一种保留模型，以更好地理解非糖基化和糖基化重链片段之间的分离选择性，利用葡聚糖-硼酸盐加合物与治疗性抗体聚糖部分之间的相互作用。单体与交联剂的比例在整体分析时间中起着重要作用，并影响非糖基化和常规（糖基化）重链片段之间的分离选择性；然而，它对单克隆抗体常规和非糖基化完整形式的分离没有影响。引入三维选择性图为手头的分离问题提供了一个简单的分离优化选项。
