Effect of Pegylation on Self-Association of IFN-α2b

Pegylation of therapeutic proteins is an established technology used to enhance the bioavailability of an active pharmaceutical ingredient in the body of patients. While the physiochemical properties of pegylated monomeric proteins have been extensively described, there is still limited information on the characterization of pegylated oligomeric proteins. In this study, we report the characterization of a pegylated interferon alpha2b (PEGIFN-alpha 2b) concentration-dependent oligomerization by a series of orthogonal biochemical and biophysical methods. These methods include sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation, Matrix-assisted laser desorption ionization, and size exclusion chromatography of bissulfosuccinimidyl suberate cross-linked PEGIFN. We report here that PEGIFN-alpha 2b self-associates in a concentration-dependent manner into mainly monomers, dimers, and trimers. In the presence of the chemical cross-linker, PEGIFN-alpha 2b is primarily monomeric (57%) at concentration lower than 0.3 mg/mL and contains about equal amount of monomers and dimers (47.0% and 37.7%, respectively), about 15% of trimers, and up to 4% of higher molecular weight species at 0.7 mg/mL and above.