Identification of the major positional isomer of pegylated interferon alpha-2b.
Interferons display a wide range of antiviral, antiproliferative, and immunomodulatory activities on a variety of cell types and have been used to treat many diseases including hairy-cell leukemia and hepatitis B and C and have also been applied to other therapeutic areas. To improve the pharmacological properties of interferon (IFN) alpha-2b, a long-acting pegylated form (PEG-IFN) has been developed [PEG, monomethoxy poly(ethylene glycol) with average molecular mass of 12 000 Da]. PEG-IFN is a mixture of pegylated proteins with differing sites of PEG attachment. To identify the major positional isomer in the pegylated material [PEG-IFN(His-34)], NMR studies were conducted on a subtilisin-digested N-acetylated peptide of the major positional isomer [PEG-IFN(His-34)dig], synthetic peptide analogues containing His-34, as well as unmodified IFN and PEG-IFN(His-34). Our studies reveal a novel interferon-polymer attachment site as a histidine-linked interferon conjugate. We show that the major component of PEG-IFN is pegylated in the imidazole side chain of histidine-34. Chemical shift data suggest that pegylation occurs mainly at the N(delta)(1) position in the imidazole side chain of this residue. This positional isomer, PEG-IFN(His-34), comprises approximately 47% of the total pegylated species when PEG-IFN is synthesized under the current experimental conditions at pH 6.5 with an electrophilic derivative of PEG, succinimidyl carbonate PEG. The reversibility of the histidine modification was examined. The PEG-imidazole adduct in the intact protein, PEG-IFN(His-34), is labile but much more stable than in the peptide, PEG-IFN(His-34)dig. Apparently, the tertiary structure of the intact protein protects the His(34)-imidazole ring from depegylation.
Wang
Y S
YS
Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USA.
Youngster
S
S
Bausch
J
J
Zhang
R
R
McNemar
C
C
Wyss
D F
DF
D004355:Drug Stability; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry* / Q000302:isolation & purification; D007536:Isomerism; D008027:Light; D019906:Nuclear Magnetic Resonance, Biomolecular; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000302:isolation & purification; D011108:Polymers / Q000737:chemistry; D011994:Recombinant Proteins; D012542:Scattering, Radiation
D007093:Imidazoles; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011108:Polymers; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D006639:Histidine; C029899:imidazole; C028210:monomethoxypolyethylene glycol; C100416:peginterferon alfa-2a