Identification of the Major Positional Isomer of Pegylated Interferon Alpha-2b

Abstract

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.

Design of homogeneous, monopegylated erythropoietin analogs with preserved in vitro bioactivity

“…ARANESP® has a 2-to 3-fold longer half-life than Epo, which permits once per week dosing in the clinical setting [5,7]. Covalent modification of proteins with polyethylene glycol (PEG) is an alternative technology that has proven useful for extending the circulating half-lives of therapeutic proteins [8][9][10][11]. PEGylated proteins often have 5-fold or greater improved half-lives relative to the unmodified protein [8][9][10][11].…”

“…Covalent modification of proteins with polyethylene glycol (PEG) is an alternative technology that has proven useful for extending the circulating half-lives of therapeutic proteins [8][9][10][11]. PEGylated proteins often have 5-fold or greater improved half-lives relative to the unmodified protein [8][9][10][11]. The most commonly used method for PEGylating proteins attaches PEG to amine groups in proteins, typically at lysine residues and/or at the N-terminal amino acid.…”

Phase 1 study of polyethylene glycol formulation of interferon α-2B (Schering 54031) in Philadelphia chromosome–positive chronic myelogenous leukemia

“…22,23 A semisynthetic formulation (protein-polymer conjugate of IFN-␣-2b) was developed by attaching a single PEG 12 000 molecule to the ⑀ amino group of selected lysine residues in the IFN-␣-2b molecule or the N-terminal amino acid. 24 PEG modification of proteins has led to the development of several PEG-proteins of current or future potential importance in therapy, including PEGasparaginase, 25,26 PEG-erythropoietin, 27 PEG-granulocyte colony stimulating factor, recombinant PEG human megakaryocyte growth and development factor, 28,29 and others. 30,31 PEG modification of proteins prolongs their plasma half-life, reduces antigenicity and immunogenicity, and reduces sensitivity to proteolysis.…”

<i>In Vivo</i> Pharmacokinetics and Pharmacodynamics of Positional Isomers of Mono-PEGylated Recombinant Human Granulocyte Colony Stimulating Factor in Rats

“…10,13) The PEGylated therapeutic proteins using mPEG-NHS are consequently heterogeneous and in the number of individual positional isomers and/or multiple isomers that could be obtained after random PEGylation. 13,14) The structural characterization of individual PEG positional isomers is a significant chemistry and manufacturing control (CMC) challenges in the manufacturing process for PEGylated therapeutic proteins. 15) We previously described the preparation of a long-acting, mono-PEGylated recombinant human granulocyte colony stimulating factor (rhG-CSF) using trimeric-structured mPEG-NHS 16) and the structural characterization of individual positional isomers isolated from mono-PEGylated rhG-CSF using a preparative two-step chromatography method developed in our laboratory.…”