Selection of migration parameters for a highly reliable assignment of bands of isoforms of erythropoietin separated by capillary electrophoresis

Lacunza, Izaskun; Lara-Quintanar, Pilar; Moya, Galina; Sanz, J.; Diez-Masa, Jose Carlos; Frutos, Mercedes de

doi:10.1002/elps.200305831

Cited by 30 publications

(28 citation statements)

References 21 publications

(33 reference statements)

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“…Different papers have demonstrated the suitability of the use of the m e instead of migration times in order to improve the method reproducibility, and assure a correct glycoform assignment [22,24]. In this work, m e have been calculated for each resolved glycoform from the migration times obtained for each glycoform and the migration time of the EOF obtained from the negative peak owing to the water present in the sample.…”

Section: Cdt Detection In Serum Samples By Ce-uvmentioning

confidence: 99%

Characterization of transferrin glycoforms in human serum by CE‐UV and CE‐ESI‐MS

et al. 2007

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Human transferrin (Tf) is a model glycoprotein for congenital disorders of glycosylation (CDG) diagnosis. In the last few years, new CE-UV methods for intact Tf glycoforms analysis have been developed using nonvolatile BGEs and organic modifiers. However, the use of these BGEs does not allow the coupling of these procedures with electrospray MS (ESI-MS). In this study, a new CE-UV separation method of Tf glycoforms is developed, using a double-layer stable coating and a volatile BGE based on ammonium acetate. The separation method is optimized using standard Tf and their potential is demonstrated applying the method to the analysis of sera Tf from healthy individuals and CDG patients. The CE-UV separation method has been coupled to ESI-MS detection. Main parameters such as sheath liquid composition are optimized in order to obtain a good sensitivity. The CE-ESI-MS method has also been used in serum samples obtaining the separation of the different proteins present in serum and partial separation of Tf glycoforms. Different mass spectra and deconvoluted molecular masses were obtained for each sialoform, allowing unequivocal glycoform identification.

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Section: Cdt Detection In Serum Samples By Ce-uvmentioning

confidence: 99%

Characterization of transferrin glycoforms in human serum by CE‐UV and CE‐ESI‐MS

et al. 2007

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“…Structural characterization of rHuEPO has been usually performed by peptide mapping after enzymatic digestion [2,[8][9][10] or glycan analysis after chemical or enzymatic release [11][12][13]. At the intact protein level, rHuEPO has been mainly analysed by CZE [14][15][16][17], IEF [18][19][20], 2-DE [21], CIEF [22] and MALDI-TOF-MS [23]. Nevertheless, the structural information provided by these methods is limited.…”

Section: Introductionmentioning

confidence: 99%

Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis – electrospray – time‐of‐flight mass spectrometry

et al. 2006

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Glycosylation of recombinant human erythropoietin (rHuEPO) is a post-translational process that alters biological activity, solubility and lifetime of the glycoprotein in blood, and strongly depends on the type of cell and the cell culture conditions. A fast and simple method providing extensive carbohydrate information about the glycans present in rHuEPO and other glycoproteins is needed in order to improve current methods in drug development or product quality control. Here, an improved method for intact rHuEPO glycoform characterization by CZE-ESI-TOF MS has been developed using a novel capillary coating and compared to a previous study. Both methods allow a fast separation in combination with accurate mass characterization of the single protein isoforms. The novel dynamic coating provides a separation at an EOF close to zero, enabling better separation. This results in an improved mass spectrometric resolution and the detection of minor isoforms. In order to assign an unequivocal carbohydrate composition to every intact glycoform, a CZE-ESI-MS separation method for enzymatically released underivatized N-glycans has been developed. The TOF MS allows the correct identification of the glycans due to its high mass accuracy and resolution. Therefore, glycan modifications such as acetylation, oxidation, sulfation and even the exchange of OH by NH(2) are successfully characterized. Information of the protein-backbone molecular mass has been combined with results from peptide analysis (revealing information about O-glycosylation) and from the glycan analysis, including the detection of as yet undescribed glycans containing four antennae and five sialic acids. This allows an unequivocal assignment of an overall glycosylation composition to the molecular masses obtained for the intact rHuEPO glycoforms.

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“…Interday reproducibility for migration times was relatively high, but the RSD values obtained for m e were four to five times lower. It has been already suggested that the iden- tification of the separated glycoforms by the methodology proposed by the EurPh may be improved using the m e or the migration time ratios (t app /t EOF ) of the glycoforms instead of their migration times (t app ) [15,30]. Figure 1 shows the electropherograms obtained for rHuEPO BRP, epoetin alpha and epoetin beta in m e scale.…”

Section: Capillary Electrophoresismentioning

confidence: 97%

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

et al. 2006

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Estimation of the composition of recombinant human erythropoietin mixtures using capillary electrophoresis and multivariate calibration methods A multivariate calibration method using partial least-squares (PLS) is proposed in order to characterize binary mixtures of two types of recombinant human erythropoietin (epoetin alpha and beta), based on the analysis of the highly overlapped UV-electrophoretic profiles obtained with the CE methodology recommended by the European Pharmacopoeia (EurPh). A two-factor PLS-1 model was developed and validated using mixtures of alpha and beta epoetins. Glycoforms were identified according to their effective electrophoretic mobility values and the normalized area values of each glycoform peak were used as multivariate data. Calibration and validation results were satisfactory. The PLS-1 model was successfully used for determination of epoetin alpha and beta contents in the rHuEPO provided by the EurPh as a biological reference product.

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Selection of migration parameters for a highly reliable assignment of bands of isoforms of erythropoietin separated by capillary electrophoresis

Cited by 30 publications

References 21 publications

Characterization of transferrin glycoforms in human serum by CE‐UV and CE‐ESI‐MS

Characterization of transferrin glycoforms in human serum by CE‐UV and CE‐ESI‐MS

Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis – electrospray – time‐of‐flight mass spectrometry

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

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