Sarkozy Daniel, Guttman Andras
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, H-4032 Debrecen, Hungary.
Translational Glycomics Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, 10 Egyetem Street, H-8200 Veszprem, Hungary.
Gels. 2022 Jan 19;8(2):67. doi: 10.3390/gels8020067.
Capillary sodium dodecyl sulfate gel electrophoresis has long been used for the analysis of proteins, mostly either with entangled polymer networks or translationally cross-linked gels. In this paper capillary agarose gel electrophoresis is introduced for the separation of low molecular weight immunoglobulin subunits. The light (LC24 kDa) and heavy (HC50 kDa) chain fragments of a monoclonal antibody therapeutic drug were used to optimize the sieving matrix composition of the agarose/Tris-borate-EDTA (TBE) systems. The agarose and boric acid contents were systematically varied between 0.2-1.0% and 320-640 mM, respectively. The influence of several physical parameters such as viscosity and electroosmotic flow were also investigated, the latter to shed light on its effect on the electrokinetic injection bias. Three dimensional Ferguson plots were utilized to better understand the sieving performance of the various agarose/TBE ratio gels, especially relying on their slope (retardation coefficient, K) value differences. The best resolution between the LC and non-glycosylated HC IgG subunits was obtained by utilizing the molecular sieving effect of the 1% agarose/320 mM boric acid composition (ΔK = 0.035). On the other hand, the 0.8% agarose/640 mM boric acid gel showed the highest separation power between the similar molecular weight, but different surface charge density non-glycosylated HC and HC fragments (ΔK = 0.005). It is important to note that the agarose-based gel-buffer systems did not require any capillary regeneration steps between runs other than simple replenishment of the sieving matrix, significantly speeding up analysis cycle time.
毛细管十二烷基硫酸钠凝胶电泳长期以来一直用于蛋白质分析，大多采用缠结聚合物网络或翻译交联凝胶。本文介绍了用于分离低分子量免疫球蛋白亚基的毛细管琼脂糖凝胶电泳。使用单克隆抗体治疗药物的轻链（LC24 kDa）和重链（HC50 kDa）片段来优化琼脂糖/Tris-硼酸盐-EDTA（TBE）系统的筛分基质组成。琼脂糖和硼酸含量分别在0.2 - 1.0%和320 - 640 mM之间系统变化。还研究了几个物理参数的影响，如粘度和电渗流，后者用于阐明其对电动进样偏差的影响。利用三维弗格森图来更好地理解各种琼脂糖/TBE比例凝胶的筛分性能，特别是依赖于它们的斜率（阻滞系数，K）值差异。通过利用1%琼脂糖/320 mM硼酸组成的分子筛效应（ΔK = 0.035），在LC和非糖基化HC IgG亚基之间获得了最佳分辨率。另一方面，0.8%琼脂糖/640 mM硼酸凝胶在相似分子量但不同表面电荷密度的非糖基化HC和HC片段之间显示出最高的分离能力（ΔK = 0.005）。需要注意的是，基于琼脂糖的凝胶缓冲系统在运行之间除了简单补充筛分基质外，不需要任何毛细管再生步骤，显著加快了分析周期时间。
