Size of Pegylated Protein Conjugates Studied by Various Methods

Pegylation as the most widely used methodology for improving biopharmaceutical drugs generates considerable changes in physicochemical and biological properties of proteins. Most of the positive pharmacological effects of pegylated proteins are related to an increased hydrodynamic volume and size, respectively. To explore the size impact of polyethyleneglycol (PEG), a series of well-defined conjugates of interferon alpha 2b (IFN) were prepared with PEGs of different lengths and shapes specifically attached to the N-terminal amino group of the protein. For characterization, various methods have been used, not only common methods for estimating molecular mass and size, such as size exclusion chromatography, electrophoretic mobility in polyacrylamide gel and dynamic light scattering, but also ion exchange and reverse phase chromatographies. Although charge and hydrophobicity based separations are not directly connected with the size of molecules, their use resulted in very interesting correlations between elution times and molecular mass of the PEG-IFN conjugates.