Purpose: The conjugation of interferon-alpha2b (IFN-alpha2b) to a branched-chain (40,000) polyethylene glycol (PEG2,40K) was studied.
Methods: We studied the conjugation of IFN-alpha2b at different pH values (6.5, 7, and 8), using the PEG2,40K reagent in either solution or solid state. MonoPEGylated interferon was isolated by ion-exchange chromatography and characterized using (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (2) cation exchange high-performance liquid chromatography, (3) bicinchoninic acid protein assay, (4) enzyme-linked immunosorbent assay, (5) cell-based bioassays, (6) thermal stability (at 60 degrees C), (7) tryptic digestion, and (8) pharmacokinetics in rats.
Results: PEGylation reaction gave 30-55% PEG2,40K-IFN-alpha2b, 1-10% polyPEGylated interferon, and 35-70% unmodified IFN-alpha2b. Compared to the polyPEGylated IFN-alpha2b species, the pure (96%) monoPEGylated conjugate retained a significantly higher bioactivity (IU/mg): 1.7x10(4)+/-8.5x10(3) vs. 2.8x10(6)+/-1.4x10(6) for antiviral and 1.9x10(4)+/-9.5x10(3) vs. 3.1x10(6)+/-1.6x10(6) for antiproliferative activity. Immunorecognition against IFN was reduced by the PEG2,40K moiety in the conjugate. This monoPEGylated IFN-alpha2b, which migrated as a single band in gel electrophoresis, was found to be a heterogeneous, complex mixture of different positional isomers. PEGylation markedly enhanced both the resistance to tryptic degradation and the thermal stability of IFN-alpha2b. The serum half-life of 40K PEG-IFN was 330-fold longer, while plasma residence time was increased 708 times compared to native IFN.
Conclusion: The PEG2,40K conjugate of IFN-alpha2b has increased in vitroand in vivo stability as compared to the native cytokine.