Characterization of N‐glycan heterogeneities of erythropoietin products by liquid chromatography/mass spectrometry and multivariate analysis

Abstract

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. 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. 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. 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.