Characterization of the self-association of human interferon-α2b, albinterferon-α2b, and pegasys.

The self-association of human interferon-α2b (hIFN-α2b), albinterferon-α2b (a recombinant protein with human serum albumin and hIFN-α2b peptides fused together in a single polypeptide chain), and Pegasys (PEGylated hIFN-α2a) was characterized by analytical ultracentrifugation analyses. By examining the apparent sedimentation coefficient distribution profiles of each protein at different concentrations, it was concluded that the above three proteins are self-associating in albinterferon-α2b formulation buffer. By model fitting of sedimentation data using SEDANAL software, the stoichiometry and equilibrium constants of the self-association of these proteins were characterized. The self-association of hIFN-α2b results in the formation of stable dimers, fast-reversible tetramers, octamers, and hexadecamers. In contrast, although both albinterferon-α2b and Pegasys are self-associated, their self-association stoichiometries are significantly different from that of hIFN-α2b. The self-association of albinterferon-α2b results in the formation of reversible dimers and trimers, whereas the self-association of Pegasys gives only reversible dimers. The self-association behaviors of hIFN-α2b and albinterferon-α2b involves attractive electrostatic forces, which can be suppressed to a negligible level in low pH (pH 4.0-4.5) and high salt concentration (400 mM NaCl) buffer, allowing quantification of their size variant contents by sedimentation velocity analysis.