Identification of the major positional isomer of pegylated interferon alpha-2b

Interferons display a wide range of antiviral, antiproliferative, and immun omodulatory activities on a variety of cell types and have been used to tre at many diseases including hairy-cell leukemia and hepatitis B and C and ha ve also been applied to other therapeutic areas. To improve the pharmacolog ical properties of interferon (IFN) alpha-2b, a long-acting pegylated form (PEG-IFN) has been developed [PEG, monomethoxy poly(ethylene glycol) with a verage molecular mass of 12 000 Da]. PEG-IFN is a mixture of pegylated prot eins with differing sites of PEG attachment. To identify the major position al isomer in the pegylated material [PEG-IFN(His-34)], NMR studies were con ducted on a subtilisin-digested N-acetylated peptide of the major positiona l isomer [PEG-IFN(His-34)dig], synthetic peptide analogues containing His-3 4, as well as unmodified IFN and PEG-IFN(His-34). Our studies reveal a nove l interferon-polymer attachment site as a histidine-linked interferon conju gate. We show that the major component of PEG-IFN is pegylated in the imida zole side chain of histidine-34. Chemical shift data suggest that pegylatio n occurs mainly at the N-delta 1 position in the imidzole side chain of thi s residue. This positional isomer, PEG-IFN(His-34), comprises approximately 47% of the total pegylated species when PEG-IFN is synthesized under the c urrent experimental conditions at pH 6.5 with an electrophilic derivative o f PEG, succinimidyl carbonate PEG. The reversibility of the histidine modif ication was examined. The PEG-imidazole adduct in the intact protein, PEG-I FN(His-34), is labile but much mon stable than in the peptide, PEG-IFN(His- 34)dig. Apparently, the tertiary structure of the intact protein protects t he His(34)-imidazole ring from depegylation.