Development of fully functional proteins with novel glycosylation via enzymatic glycan trimming

Recombinant glycoproteins present unique challenges to biopharmaceutical development, especially when efficacy is affected by glycosylation. In these cases, optimizing the protein's glycosylation is necessary, but difficult, since the glycan structures cannot be genetically encoded, and glycosylation in nonhuman cell lines can be very different from human glycosylation profiles. We are exploring a potential solution to this problem by designing enzymatic glycan optimization methods to produce proteins with useful glycan compositions. To demonstrate viability of this new approach to generating glycoprotein-based pharmaceuticals, the N-linked glycans of a model glycoprotein, ribonuclease B (RNase B), were modified using an alpha-mannosidase to produce a new glycoprotein with different glycan structures. The secondary structure of the native and modified glycoproteins was retained, as monitored using circular dichroism. An assay was also developed using an RNA substrate to verify that RNase B had indeed retained its function after being subjected to the necessary glycan modification conditions. This is the first study that verifies both activity and secondary structure of a glycoprotein after enzymatic glycan trimming for use in biopharmaceutical development methods. The evidence of preserved structure and function for a modified glycoprotein indicates that extracellular enzymatic modification methods could be implemented in producing designer glycoproteins.