Structural and biophysical characterization of the 40 kDa PEG-interferon-α2a and its individual positional isomers

The human recombinant Interferon-alpha(2a) (IFN alpha(2a)) is a potent drug (Roferon-A) to treat various types of cancer and viral diseases including Hepatitis B/C infections. To improve the pharmacological properties of the drug, a new pegylated form of IFN alpha(2a) was developed (PEGASYS). This 40 kDa PEG-conjugated IFN alpha(2a) ((40)PEG-IFN alpha(2a)) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side chain of IFN alpha(2a). (40)PEG-IFN alpha(2a) is a mixture of mainly six monopegylated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively. Here we report the detailed structural and biophysical characterization of (40)PEG-IFNU alpha(2a) and its positional isomers, in comparison with IFN alpha(2a), using NMR spectroscopy, analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. Our results show that the three-dimensional structure of IFN alpha(2a). is not modified by the presence of the polymer in all positional isomers constituting (40)PEG-lFN alpha(2a). Regardless of where the PEG-polymer is attached, it adopts a very mobile and flexible random coil conformation, producing a shield for the protein without a permanent coverage of the protein surface. Hydrodynamic data indicate that the protein-attached PEG has a slightly more compact random-coil structure than the free PEG-polymer. Our results also provide evidence of significant structural and physicochemical advantages conferred by the pegylation: increase of the effective hydrodynamic volume and modification of the molecular shape, higher temperature stability, and reduced tendency for aggregation. These results are of tremendous pharmacological interest and benefit as was clinically shown with PEGASYS. This study constitutes a new standard for the characterization of pegylated proteins and enables an important step toward the understanding on a molecular level of the binding of (40)PEG-IFN alpha(2a), and its positional isomers to its cellular receptors.