PEGylated interferon-alpha2b: a branched 40K polyethylene glycol derivative.
OBJECTIVE
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.


CONCLUSIONS
The PEG2,40K conjugate of IFN-alpha2b has increased in vitroand in vivo stability as compared to the native cytokine.
Ramon
Jose
J
Formulation Development Department, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
Saez
Vivian
V
Baez
Reynier
R
Aldana
Raymersy
R
Hardy
Eugenio
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000998:Antiviral Agents / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005079:Excipients; D006358:Hot Temperature; D006868:Hydrolysis; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology; D008297:Male; D010449:Peptide Mapping; D011092:Polyethylene Glycols / Q000737:chemistry*; D011804:Quinolines / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins; D014357:Trypsin / Q000737:chemistry
D000911:Antibodies, Monoclonal; D000998:Antiviral Agents; D005079:Excipients; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011804:Quinolines; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; C047117:bicinchoninic acid; D014357:Trypsin