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PEGylated interferon-α2b: A branched 40K polyethylene glycol derivative

Abstract:

Purpose. The conjugation of interferon-α2b (IFN-α2b) to a branched-chain (40,000) polyethylene glycol (PEG2,40K) was studied. Methods. We studied the conjugation of IFN-α2b 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°C), (7) tryptic digestion, and (8) pharmacokinetics in rats. Results. PEGylation reaction gave 30-55% PEG2,40K-IFN-α2b, 1-10% polyPEGylated interferon, and 35-70% unmodified IFN-α2b. Compared to the polyPEGylated IFN-α2b species, the pure (96%) monoPEGylated conjugate retained a significantly higher bioactivity (IU/mg): 1.7 × 104 ± 8.5 × 103 vs. 2.8 × 106 ± 1.4 × 106 for antiviral and 1.9 × 104 ± 9.5 × 103 vs. 3.1 × 106 ± 1.6 × 106 for antiproliferative activity. Immunorecognition against IFN was reduced by the PEG2,40K moiety in the conjugate. This monoPEGylated IFN-α2b, 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-α2b. 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-α2b has increased in vitroand in vivo stability as compared to the native cytokine. © 2005 Springer Science + Business Media, Inc.