Isolation, structural characterization, and antiviral activity of positional isomers of monopegylated interferon alpha-2a (PEGASYS).
Interferon alpha-2a plays an essential role in the treatment of chronic hepatitis C, but it is limited in its efficacy by the short in vivo half-life. To improve the half-life and efficacy, interferon alpha-2a is conjugated with a 40-kDa branched polyethylene glycol moiety (PEG-IFN, PEGASYS). From this preparation the positional PEG-IFN isomers were isolated and characterized by different analytical methods and antiviral assay. Two chromatographic steps were used to separate and purify nine isomers. The analytical methods IE-HPLC, RP-HPLC, SE-HPLC, SDS-PAGE, and MALDI-TOF MS indicated that each of these nine isomers is conjugated to the branched polyethylene glycol chain at a specific lysine. No isomer with a modification at the amino terminus was observed. All positional isomers induced viral protection of MDBK cells in the antiviral assay. When comparing the quantitative potency of the individual isomers with the whole mixture of PEG-IFN, significant differences in the specific activities were observed: PEG-Lys(31) and PEG-Lys(134) showed higher activities than the mixture, PEG-Lys(164) was equal to the mixture, whereas the activities of PEG-Lys(49), PEG-Lys(70), PEG-Lys(83), PEG-Lys(112), PEG-Lys(121), and PEG-Lys(131) were lower.
Foser
Stefan
S
Department of Pharma Technical Operations Biotechnology, Biotech Products, Biotech Development and Production, Hoffmann-La Roche Ltd., Building 066/508, Ch-4070 Basel, Switzerland.
Schacher
Alfred
A
Weyer
Karl A
KA
Brugger
Doris
D
Dietel
Elke
E
Marti
Stefan
S
Schreitmüller
Thomas
T
D000998:Antiviral Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D007536:Isomerism; D013058:Mass Spectrometry; D008958:Models, Molecular; D015394:Molecular Structure; D010449:Peptide Mapping; D011092:Polyethylene Glycols; D011487:Protein Conformation; D011994:Recombinant Proteins
D000998:Antiviral Agents; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; C417083:peginterferon alfa-2b; C100416:peginterferon alfa-2a