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PEGylated Interferon-α2b: A Branched 40K Polyethylene Glycol Derivative

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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.

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Abbreviations

BCA:

bicinchoninic acid

CBB:

Coomassie brilliant blue

ELISA:

enzyme-linked immunosorbent assay

IEC:

ion-exchange chromatography

IFN:

interferon

mAb:

monoclonal antibody

Mr:

average molecular mass

PEG:

polyethylene glycol

RP-HPLC:

reversed-phase high performance liquid chromatography

SDS-PAGE:

sodium dodecylsulfate-polyacrylamide gel electrophoresis

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Acknowledgments

We thank our colleagues from the Department of Pharmacology and Toxicology of the CIGB (Havana, Cuba) and the Center of Biological Evaluation and Research of the Institute of Pharmacy and Foods (Havana University) for their invaluable technical assistance and instructive discussions. Jose Luis Fernandez and Alfredo Pereira are thanked for a final edit of the language in the revised version. We also acknowledge Prof. C. H. Fox for his interest and encouragement.

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Authors and Affiliations

  1. Formulation Development Department, Center for Genetic Engineering and Biotechnology, Havana, Cuba

    Jose Ramon, Vivian Saez, Reynier Baez & Raymersy Aldana

  2. Vaccine Department, Center for Genetic Engineering and Biotechnology, Havana, Cuba

    Eugenio Hardy

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  1. Jose Ramon
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Correspondence to Eugenio Hardy.

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Ramon, J., Saez, V., Baez, R. et al. PEGylated Interferon-α2b: A Branched 40K Polyethylene Glycol Derivative. Pharm Res 22, 1375–1387 (2005). https://doi.org/10.1007/s11095-005-5278-4

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