Two-peak Elution Profile of a Bispecific VHH-IgG Fusion Protein in Ion Exchange Purification Process caused by Atypical N-Glycosylation

In-depth analysis and thorough understanding of Nglycosylation is critical for therapeutic proteins. For IgG or IgG-like molecules, the conserved N-glycosylation at Asn297 has critical safety and efficacy implications and therefore is under close scrutiny. Any additional N-glycosylation that increases complexity and heterogeneity of the molecule, should be avoided at the early stage of drug discovery. However, unexpected glycosylation at atypical sites brings unforeseen challenges for antibody drug development process. Here, we observed a two-peak elution profile when developing ion exchange purification process for a novel bispecific VHH-IgG fusion protein. The charge and size properties of the two peaks were characterized by a variety of analytical methods. Two atypical N-glycosylation sites in the VHH domain were identified by means of mass spectrometry and confirmed by site-specific mutagenesis. Further characterization indicated that G0F was the predominant glycan species for both sites with varying occupancy. Interestingly, the addition of neutral glycans changed the charge behaviour of the fusion protein, leading to the unexpected ion exchange profile. These findings suggest that glycosylation may occur at non-canonical sites and may have significant effect on the process development and drug developability.