Mass spectrometry-based characterization of acidic glycans on protein therapeutics

Glycosylation is a critical posttranslational modification that affects the physiochemical and biological properties of glycoproteins. Specifically, acidic glycans such as phosphorylated, sialylated and sulfonated glycoforms have well known biological implications; therefore, a detailed understanding of these structures is essential for defining the attributes of therapeutic glycoproteins. The clinical significance of these attributes has increased the analytical requirements for development of therapeutic glycoproteins. Despite current advances in mass-spectrometric methodologies, challenges remain in characterizing these heterogeneous modifications. Here we present the global and site-specific characterization of acidic N-linked glycans from three therapeutic enzymes, velaglucerase alfa, idursulfase and agalsidase alfa. Orthogonal methodologies, including MALDI-TOF MS, static ESI-MS/MS, RPLC–MS, and HILIC–MS, are used to characterize N-linked glycans to support the development of enzyme replacement therapeutics. In addition, the results of these studies can provide identification, structural elucidation, and quantitation of the different acidic glycoforms that is often used to understand the glycan composition of these molecules during product development.

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► Several mass spectrometric techniques were required to characterize the diverse nature of phosphorylated, sialylated and sulfonated N-glycans on several therapeutic glycoproteins.
► LC-MS intact protein analysis and MALDI analysis of the total released glycans provided a qualitative assessment of the type of N-linked glycans present on each glycoprotein.
► The detailed structural characterization of the released phosphorylated glycans reveals the position of the phosphate as well as isomeric forms of the mannose residues.
► Site-specific LCMS quantitation of glycopeptides is highly dependent upon the acidic nature of the glycan(s).
► Unique sulfonated sialyl N-linked glycans were identified and characterized through HILIC-MS methods