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

Abstract

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.

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

“…4,8,10,43,44 These modifications were mainly observed in highly sialylated glycans and include acetylation of sialic acids, N-acetylneuraminic (Neu5Ac) / N-glycolylneuraminic acid (Neu5Gc) variation, and elongation of the glycan chains because of the presence of LacNAc repeats. Up to three modifications occurred per glycan.…”

“…This phenomenon has not been extensively reported and there are only a few studies in the literature dealing with the presence of Neu5Gc in rHuEPO. 8,10,44,53,60 Even though Neu5Gc is a common sialic acid in most mammals, it has been demonstrated to be absent in healthy humans and only small amounts have been found in some tumors and meconium. 61 The CE-MS method succeeded in detecting this heterogeneity of acidic monosaccharides without removing the sialic acid from the glycan chain by acid hydrolysis as it has been commonly reported.…”

A multiway approach for classification and characterization of rabbit liver apothioneins by CE‐ESI‐MS

“…5A and B). The accuracy of the Mm values obtained with MCR-ALS or PARAFAC will have important implications when an reliable identification is necessary, and it could be improved by using an MS detector with improved mass accuracy and resolution [9]. However, at this point, it is important to emphasize the excellent accuracy of the Mm data obtained from both multivariate data analysis models.…”

“…Several CE-ESI-MS have been described for the selective separation and characterization of protein isoforms [1-4, 6-7, 9]. However, the performance of CE-ESI-MS is limited and resolution problems could arise when handling complex mixtures of protein isoforms, such as human erythropoietin, which is a mixture of around 100 glycoforms [9]. In such cases, the methods traditionally used for the analysis of MS data may be excessively time consuming.…”

Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity

“…19 Intact mass of resolved peaks can reveal differential glycan forms, but additional processing and data analysis (e.g., tryptic peptide mapping) is required to identify the location and nature of specific chemical modifications (e.g., residue specific deamidation). 20,21 Thus, a rapid and simple separation method with greater information content would be highly beneficial.…”