Fractionation of glycoforms of recombinant human erythropoietin by preparative capillary isotachophoresis

Abstract

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.

Some possibilities of an analysis of complex samples by a mass spectrometry with a sample pretreatment by an offline coupled preparative capillary isotachophoresis

“…3. The details of fractionation procedure are given in the paper of Madajová et al [50]. The experiments without the injection of the analyte served as the reference.…”

“…Model matrix in our study was urine which naturally contains high concentration of sodium cation (the concentration level is about 0.1 mol/L) and, therefore, sodium cation at 10 mmol/L concentration was used as the leading ion and acetic acid (serving as the counterion) was used for adjusting the pH of the LE to 5.00. b-Alanine at 20 mmol/L concentration was used as the terminating ion and acetic acid was used as the counterion. For the isolation of analyte from the potential interfering constituents originating from the complex ionic matrix in pITP, it is wise to use the discrete spacers [50] which can divide the mobility span interval between the leading and terminating ions into the given discrete and well-defined narrower mobility spans. The proper mixture of discrete spacers for the isolation of buserelin from the urine matrix was found consisting of five constituents (see Section 2) which divided the mobility span interval between the leading and terminating ions into six parts.…”

“…The main advantages of this technique are: (i) rapid simplification and/or reduction of complex ionic matrices; (ii) well-defined sample pretreatment conditions in ITP separation mode; (iii) isolation of analytes into well-defined fraction with almost known composition when discrete spacer technique is used [50]; (iv) easily obtainable compatibility of the whole analytical separation system when non-CE analytical techniques are used. In 1981, Kenndler and Kaniansky published the first article dealing with the combination of electrophoretic technique and mass spectrometry.…”

“…The works [40-42, 44, 45] demonstrated the need of using the column-coupling technique [46] in the cases when the analyte is present in the complex mixture(s) which was successfully shown for the combination of pITP with different analytical techniques [47][48][49][50]. The benefits of column-coupling pITP before the combination of gas chromatography with mass spectrometric (GC-MS) or atomic emission detection (GC-AED) on the identification and quantitative determination of prometryne in the plant extracts [51] were clearly proved.…”

Estimation of the composition of recombinant human erythropoietin mixtures using capillary electrophoresis and multivariate calibration methods

“…Several CE methodologies have been described for the separation of rHuEPO glycoforms [11][12][13][14][15][16][17][18]. Recently, it has been demonstrated that accurate mass measurements of intact rHuEPO glycoforms are possible using CE coupled online with a last-generation TOF mass spectrometer [14].…”