Cifuentes A, Moreno-Arribas M V, de Frutos M, Díez-Masa J C
Institute of Organic Chemistry (CSIC), Madrid, Spain.
J Chromatogr A. 1999 Jan 15;830(2):453-63. doi: 10.1016/s0021-9673(98)00875-9.
The influence of several operation conditions on separation of recombinant human erythropoietin glycoforms by capillary isoelectric focusing (cIEF) is explored. From this study it is deduced that in order to separate several glycoforms of erythropoietin, urea has to be added to sample, which should not be completely depleted of the excipients used in its formulation. On-line desalting does not provide separation enhancement for samples with high content of salt. Better resolution is obtained using a mixture of a broad and a narrow pH-range carrier ampholytes than with either one used separately. Under the experimental conditions, focusing voltages of 25 kV improve separation compared to lower and higher electric fields. Focusing times shorter than the time necessary for electric current to reach a minimum provide similar separations than longer focusing times at which a minimum value of the current has already been achieved. The optimized method allows the separation and quantitation in 12 min of at least seven bands containing glycoforms of recombinant erythropoietin with apparent isoelectric points in the range 3.78-4.69. Compared to flat-bed isoelectric focusing, cIEF provides better separation of bands of glycoforms in a shorter time, and allows quantitative determination. Capillary zone electrophoresis (CZE) gives rise to resolution of erythropoietin glycoforms similar to that obtained by cIEF. Although CZE requires a longer analysis time, its reproducibility in terms of peak area of glycoforms is better than in cIEF.
探讨了几种操作条件对毛细管等电聚焦（cIEF）分离重组人促红细胞生成素糖型的影响。通过这项研究推断，为了分离促红细胞生成素的几种糖型，必须向样品中添加尿素，且样品不应完全去除其制剂中使用的辅料。在线脱盐对于高盐含量的样品并不能提高分离效果。与单独使用宽pH范围或窄pH范围的载体两性电解质相比，混合使用这两种载体两性电解质可获得更好的分离效果。在实验条件下，与较低和较高电场相比，25 kV的聚焦电压可改善分离效果。聚焦时间短于电流达到最小值所需的时间，与电流已达到最小值时的较长聚焦时间相比，分离效果相似。优化后的方法可在12分钟内分离并定量至少七条含有重组促红细胞生成素糖型的条带，其表观等电点范围为3.78 - 4.69。与平板等电聚焦相比，cIEF能在更短时间内更好地分离糖型条带，并可进行定量测定。毛细管区带电泳（CZE）对促红细胞生成素糖型的分离效果与cIEF相似。虽然CZE需要更长的分析时间，但其糖型峰面积的重现性优于cIEF。
