[Structural Biological Approach to Biopharmaceuticals]

Detailed structural characterization of protein biopharmaceuticals is a critical step in research and development; however, this step is often hampered by the structural complexities associated with glycosylation. Most protein biopharmaceuticals are modified with structurally heterogeneous and dynamic oligosaccharides which govern the physicochemical properties, functionality, pharmacokinetics, and potential pathogenicity of these glycoproteins. Considering this, we have developed a structural biological approach to describe the dynamic three-dimensional structures and interactions of glycoproteins as biopharmaceuticals. We developed an NMR technique assisted by metabolic stable-isotope labeling that can provide useful atomic-level probes for detecting and characterizing structural perturbations of glycoproteins caused by alterations in solution conditions and production protocols, as well as by mutagenesis. We have applied this method in conjunction with X-ray crystallography to investigate the structural impacts of varying glycoforms of the Fc region of immunoglobulin G (IgG), thereby elucidating the functional roles of the Fc glycans. In particular, we have successfully elucidated the structural mechanisms by which defucosylation of the IgG-Fc region increases its affinity for Fcγ receptor IIIa, leading to an improvement in ameliorating antibody-dependent cell-mediated cytotoxicity. In addition, we applied our stable-isotope-assisted NMR method to analyzing biomolecular interactions in serum environments, which are characterized by molecular crowding and promiscuous intermolecular interactions. An integrative structural biological approach combining NMR spectroscopy, X-ray crystallography, neutron scattering, atomic force microscopy, and molecular dynamics simulation will provide new research tools that will enable the visualization of dynamic structures and interactions of glycoproteins of pharmaceutical interest, thereby providing valuable insights for the development of biopharmaceuticals.