Evaluation of protein surface modifications and their impacts on intermolecular protein-protein interactions

In this work, we explore how modifications of the protein surface can lead to changes to intermolecular protein-protein interactions. We examined how the glycosylation of gamma 1 isotype immunoglobulin (IgG1), and the covalent attachment of polyethylene glycol (PEG) to the surface of human γD-crystallin (HGD), affect inter-protein interactions and the subsequent aggregation propensity and viscosity of protein solutions respectively. IgG1 molecules have N-linked glycans with huge variability in glycan composition. Through dynamic light scattering and liquid-liquid phase boundary measurements, the impact of the IgG1 Fc N-glycan galactose content on interprotein interactions was probed, with no significant impact reported for two different IgG1 molecules. Removal of the terminal sialic acid from Fab N-glycans resulted in a significant reduction to measured kD values and an increase to Tph, indicating a shift to more attractive interprotein interactions upon sialic acid removal. We further examined how the galactose content of the Fc N-glycans affect IgG1 unfolding behaviour by nano-DSF, with no measurable effect on unfolding behaviour for IgG1 in the conditions probed here. Subsequent thermal unfolding experiments in the presence of various excipients across a concentration range also revealed no significant difference in unfolding behaviour for different IgG1 glycoforms. The impact on intermolecular interactions of the conjugation of various molecular weights of polyethylene glycol (PEG) to the surface of human γD crystallin (HGD) were also investigated. Extrapolation of B22 values from static light scattering revealed a direct link between the mass of conjugated PEG and the magnitude of a repulsive interaction introduced by PEGylation. Dynamic light scattering revealed significant decreases to diffusional mobility for molecules with higher PEG molecular weights, and an associated increase to hydrodynamic interactions were also observed. This decreased diffusional mobility and increased hydrodynamic interactions coincided with an increased viscosity for 5 kDa and 10 kDa PEGylated HGD, with the possibility of polymer entanglement also observed at the highest 10 kDa PEGylated HGD concentrations.