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RATIONALE Glycan heterogeneity on recombinant human erythropoietin (rEPO) product is considered to be one of the critical quality attributes, and similarity tests of glycan heterogeneities are required in the manufacturing process changes and developments of biosimilars. A method for differentiating highly complex and diverse glycosylations is needed to evaluate comparability and biosimilarity among rEPO batches and products manufactured by different processes. METHODS The glycan heterogeneities of nine rEPO products (four innovator products and five biosimilar products) were distinguished by multivariate analysis (MVA) using the peak area ratios of each glycan to the total peak area of glycans in mass spectra obtained by liquid chromatography/mass spectrometry (LC/MS) of N-glycans from rEPOs. RESULTS Principal component analysis (PCA) using glycan profiles obtained by LC/MS proved to be a useful method for differentiating glycan heterogeneities among nine rEPOs. Using PC values as indices, we were able to visualize and digitalize the glycan heterogeneities of each rEPO. The characteristic glycans of each rEPO were also successfully identified by orthogonal partial least-squares discrimination analysis (OPLS-DA), an MVA method, using the glycan profile data. CONCLUSIONS PCA values were useful for evaluating the relative differences among the glycan heterogeneities of rEPOs. The characteristic glycans that contributed to the differentiation were also successfully identified by OPLS-DA. PCA and OPLS-DA based on mass spectrometric data are applicable for distinguishing glycan heterogeneities, which are virtually indistinguishable on rEPO products. Copyright © 2014 John Wiley & Sons, Ltd.

The Lewis x structure [Le x , Galβ1-4(Fucα1-3)GlcNAc] motif is one of the tumor antigens and plays an important role in oncogenesis, development, cellular differentiation and adhesion. The detection of Le x -carbohydrates and their structural analysis are necessary to clarify the role of Le x in several biological events. Mass spectrometry has been preferably used for the structural analysis of carbohydrates. Especially, collision-induced dissociation (CID) tandem mass spectrometry (MS/MS), which causes a glycosidic bond cleavage, is used for carbohydrate sequencing. However, Le x cannot be identified by MS/MS due to the existence of the positional isomers, such as Lewis a [Galβ1-3(a1-4Fuc)GlcNAc]. In the present study, we demonstrate the specific detection of Le x -carbohydrates in a biological sample by using multiple-stage MS/MS (MS"). Using pyridylaminated oligosaccharides bearing Le x , we found that the Le x -motif yields a cross-ring fragment by the cleavage of a bond between C-3 and C-4 of GlcNAc in Gal(Fuc)GlcNAc. The Le x -specific cross-ring fragment ion at m/z 259 was effectively detected by sequential scans, consisting of a full MS 1 scan, data-dependent CID MS 2 scan, MS 3 of [Gal(Fuc)GlcNAc+Na] + at m/z 534, and MS 4 of [GalGlcNAc+Na] + at m/z 388. The sequential scan was applied to N-linked oligosaccharide profiling using a LC/ESI-MS n system equipped with a graphitized carbon column. We successfully detected the Le x -motif and elucidated the structures of several Le x and Lewis y [(Fucα1-2)Gal/β1-4(Fucα1-3)GlcNAc] oligosaccharides in the murine kidney used as a model tissue. Our method is expected to be a powerful tool for the specific detection of the Le x -motif, and structural elucidation of Le x -carbohydrates in biological samples. Copyright ( 2005 John Wiley & Sons, Ltd.