Combination of large volume sample stacking and reversed pH junction in capillary electrophoresis for online preconcentration of glycoforms of recombinant human erythropoietin.
Resolution of glycoforms of a glycoprotein is usually a challenge for an analytical chemist, which is often associated with the difficulty of detecting a large number of glycoforms with sufficient sensitivity. CE is the main workhorse for the analysis of glycoproteins; however, the current methods are only applicable to high concentration samples. Therefore, the development of online preconcentration approaches is important for the CE analysis of glycoproteins. In this study, we present a combined strategy for online preconcentrating glycoforms of glycoproteins, which couples two individual online preconcentration techniques, i.e., large volume sample stacking (LVSS) and reversed pH junction (RPHJ). LVSS allows for compressing a large injected sample volume into a narrow sample zone, contributing to the main sensitivity improvement, while RPHJ coordinates the preconcentration process to improve the resolution. This strategy was verified with recombinant human erythropoietin (rhEPO), a typical glycoprotein, as the test analyte, and the effects of experimental conditions were investigated. It was found that there is a compromise between sensitivity enhancement and resolution for online preconcentration of glycoprotein glycoforms. By using this strategy, the detection sensitivity can be improved by 50-100 times for rhEPO.
Liu
Jing
J
Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China.
Liu
Zhen
Z
Kang
Mingchao
M
Liu
Sichang
S
Chen
Hong-Yuan
HY
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019075:Electrophoresis, Capillary / Q000379:methods*; D004921:Erythropoietin / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009862:Online Systems; D009994:Osmolar Concentration; D011994:Recombinant Proteins
D011994:Recombinant Proteins; D004921:Erythropoietin