Fractionation of glycoforms of recombinant human erythropoietin by preparative capillary isotachophoresis.
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.
Madajová
Vlasta
V
Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia.
Simunicová
Eva
E
Kaniansky
Dusan
D
Marák
Jozef
J
Zelenská
Viera
V
D005591:Chemical Fractionation / Q000379:methods; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D004921:Erythropoietin / Q000302:isolation & purification*; D005240:Feasibility Studies; D006020:Glycopeptides / Q000302:isolation & purification*; D006031:Glycosylation; D006801:Humans; D011994:Recombinant Proteins; D015203:Reproducibility of Results
D006020:Glycopeptides; D011994:Recombinant Proteins; D004921:Erythropoietin