Madajová Vlasta, Simunicová Eva, Kaniansky Dusan, Marák Jozef, Zelenská Viera
Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia.
Electrophoresis. 2005 Jun;26(13):2664-73. doi: 10.1002/elps.200500044.
This feasibility study deals with the use of preparative capillary isotachophoresis (CITP), operating in a discontinuous fractionation mode, to the separations and isolations of glycoforms of recombinant human erythropoietin (rhEPO). The preparative CITP separations were monitored by capillary zone electrophoresis (CZE) with a hydrodynamically closed separation unit. Such a CZE system, suppressing fluctuations of the migration data linked with fluctuations of EOF and hydrodynamic flow, made possible to evaluate and compare the preparative CITP separations performed within a longer time frame. Preparative CITP, carried out in the separation unit with coupled columns of enhanced sample loadability, separating 100 microg of rhEPO in a run lasting ca. 30 min, gave the production rate higher than 55 ng/s for the rhEPO glycoforms. The preparative separations included valve isolations of the glycoforms from the ITP stack into four or six fractions. Such numbers of the fractions corresponded to typical numbers of the major glycoform peaks as resolved in CZE of rhEPO. With respect to close effective mobilities of the glycoforms and a multicomponent nature of rhEPO, the fractions contained mixtures of glycoforms with the dominant glycoforms enriched 10-100-fold, relative to the original rhEPO sample.
本可行性研究涉及使用以不连续分级模式运行的制备型毛细管等速电泳（CITP）来分离和纯化重组人促红细胞生成素（rhEPO）的糖型。制备型CITP分离过程通过带有流体动力学封闭分离单元的毛细管区带电泳（CZE）进行监测。这样的CZE系统抑制了与电渗流（EOF）和流体动力流波动相关的迁移数据波动，使得评估和比较在较长时间内进行的制备型CITP分离成为可能。在具有增强样品负载能力的耦合柱的分离单元中进行的制备型CITP，在约30分钟的运行时间内分离100微克rhEPO，rhEPO糖型的生产率高于55纳克/秒。制备型分离包括将糖型从ITP柱堆中通过阀分离成四个或六个馏分。这些馏分的数量对应于rhEPO的CZE中解析出的主要糖型峰的典型数量。鉴于糖型的有效迁移率相近以及rhEPO的多组分性质，这些馏分包含糖型混合物，其中占主导的糖型相对于原始rhEPO样品富集了10至100倍。
