Characterizing a novel hyposialylated erythropoietin by intact glycoprotein and glycan analysis

• Published in: Journal of pharmaceutical and biomedical analysis 2022-05, Vol.213, p.114686-114686, Article 114686
• Main Authors: García-Artalejo, Judey Aymed, Mancera-Arteu, Montserrat, Sanz-Nebot, Victòria, Rodríguez, Teresita, Giménez, Estela
• Format: Article
• Language: English
• Subjects: Erythropoietin; Erythropoietin - chemistry; Glycans; Glycoforms; Glycoproteins - chemistry; Humans; IEF; Mass spectrometry; Mass Spectrometry - methods; NeuroEPO plus; Polysaccharides - analysis; Recombinant Proteins - chemistry; ZIC-HILIC
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2022.114686

NeuroEPO plus is a recently developed recombinant human erythropoietin (rhEPO) without erythropoietic activity and shorter plasma half-life due to its low sialic acid content. This novel rhEPO product is under investigation as therapeutic protein in the treatment of neurodegenerative diseases owing to its neuroprotective and neurodegenerative properties. In this study, an in-depth characterization of NeuroEPO plus N-glycans was performed by a glycan isotope [12C6]/[13C6] coded aniline labeling strategy followed by capillary zwitterionic hydrophilic interaction liquid chromatography-mass spectrometry (CapZIC-HILIC-MS). A superior amount of low sialylated glycans and less branched structures were detected in NeuroEPO plus compare to other commercial rhEPOs. At the intact glycoprotein level, NeuroEPO plus glycoforms were separated by capillary zone electrophoresis with ultraviolet detection (CE-UV), optimizing the composition and pH of the separation electrolyte. Moreover, an isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) method was also optimized for the simultaneous analysis of this basic rhEPO and conventional acidic rhEPO products. The proposed glycomic and intact glycoprotein methods provide a robust and reliable analytical platform for NeuroEPO plus characterization and for its future implementation as biopharmaceutical in neurodegenerative diseases. [Display omitted] •In-depth NeuroEPO plus characterization using both intact protein and glycan analysis.•Decreased sialylation and branching could explain NeuroEPO plus neuroprotective effects.•The developed methods provide an excellent analytical platform for quality control.•The CE-UV method could be implemented by the Eur.Ph. for other highly basic rhEPOs.