Heterogeneous glycoform separation by process chromatography: I: Monomer purification and characterization

• Published in: Journal of chromatography. A Vol. 1404; p. 51
• Main Authors: Li, Yi, Xu, Xuankuo, Shupe, Alan, Yang, Rong, Bai, Kevin, Das, Tapan, Borys, Michael C, Li, Zheng Jian
• Format: Journal Article
• Language: English
• Published: Netherlands 24.07.2015
• Subjects: Chemistry Techniques, Analytical - methods; Chromatography; Glycosylation; Hydrophobic and Hydrophilic Interactions; Molecular Weight; N-Acetylneuraminic Acid - chemistry; Proteins - chemistry; Antibody purification platform; Hydrophobic interaction chromatography; Biophysical characterization; Glycosylation; Heterogeneous glycoform profile; Ion-exchange chromatography
• ISSN: 1873-3778
• DOI: 10.1016/j.chroma.2015.05.054

Fc fusion proteins with high and low sialylation were purified and separated by preparative ion-exchange and hydrophobic interaction chromatography. Heterogeneity in sialylation and glycosylation led to variation in surface charge and hydrophobicity, and resulted in multiple distinct glycoform populations in response to various purification conditions. Monomer with high sialic acid content has higher surface charge and adsorbs stronger to ion-exchange resin, while the less sialylated monomer interacts more favorably with hydrophobic resin. Extensive biophysical characterization was carried out for purified monomers at different level of sialylation. In general, different monomeric glycoforms have different surface charge and hydrophobicity, different thermal stability, and different aggregation propensity. The surface charge corresponds well with sialic acid content, as evidenced by electrophoresis, N-link domain analysis, and zeta potential results. The sialylation also contributes to minor modification of protein size, molecular mass and tertiary structure. Notably, fluorescence emission spectra and thermal transition became less distinguishable when the monomers containing low and high sialic acid were prepared in high ionic strength solution. Such finding reiterates the fact that the electrostatic forces, which are largely dependent on sialic acid content of protein, plays a dominant role in many intra- and inter-molecular interactions. Overall, the characterization data agreed well with separation behaviors and provided valuable insight to control of glycoform profile in purification process.