Stephen Kenneth Youngster

Structural and biological characterization of pegylated recombinant interferon alpha-2b and its therapeutic implications

The type I interferon alpha family consists of small proteins that have clinically important anti-infective and anti-tumor activity. Interferon alpha-2b (Intron A) combination therapy with ribavirin is the current standard of care for the treatment of chronic hepatitis C virus infection. A drawback to the therapy however, is the short serum half-life and rapid clearance of the interferon alpha protein. Schering-Plough has developed a semi-synthetic form of Intron A by attaching a 12-kDa mono-methoxy polyethylene glycol to the protein (PEG Intron) which fulfills the requirements of a long-acting interferon alpha protein while providing significant clinical benefits. A detailed physicochemical and biological characterization of PEG Intron revealed its composition of pegylated positional isomers and the specific anti-viral activity associated with each of them. Though pegylation appeared to decrease the specific activity of the interferon alpha-2b protein, the potency of PEG Intron, independent of protein concentration, was comparable to the Intron A standard at both the molecular and cellular level. Importantly, PEG Intron has demonstrated an enhanced pharmacokinetic profile in both animal and human studies. Recently, PEG Intron in combination with ribavirin has been shown to be very effective in reducing hepatitis C viral load and maintaining effective sustained viral suppression in patients. Because of the improved clinical benefits, it is anticipated that the PEG Intron plus ribavirin combination therapy will become the new standard of care for the treatment of chronic hepatitis C.

Structural and Biologic Characterization of Pegylated Recombinant IFN-α2b

The type I interferon-α (IFN-α) family is a family of natural small proteins that have clinically important anti-infective and antitumor activity. We have developed a semisynthetic protein-polymer conjugate of IFN-α2b (Intron® A) by attaching a 12,000-Da monomethoxypolyethylene glycol (PEG-12000) polymer to the protein. PEG conjugation is thought to increase the serum half-life and thereby prolong patient exposure to IFN-α2b without altering the biologic potency to the protein. Matrix-assisted laser desorption ionization/mass spectrometry (MALDI-MS), high-performance size exclusion chromatography (HPSEC), circular dichroism (CD) analysis and tryptic digestion peptide analysis of PEG Intron demonstrated that the IFN-α2b protein was approximately 95% monopegylated and that the primary, the secondary, and the tertiary structures were unaltered. Pegylation did not affect the epitope recognition of antibodies used for Intron A quantitation. An extensive analysis of the pegylated positional isomers revealed tha...

Structure, biology, and therapeutic implications of pegylated interferon alpha-2b.

Derivatization of protein-based therapeutics with polyethylene glycol (pegylation) can often improve pharmacokinetic and pharmacodynamic properties of the proteins and thereby, improve efficacy and minimize dosing frequency. This review will provide an overview of pegylation technology and pegylated protein-based drugs being used or investigated clinically. The novel therapeutic, PEG Intron(R), formed by attaching a 12-kDa mono-methoxy polyethylene glycol (PEG) to the interferon alpha-2b protein, will be discussed in detail in terms of its structure, biological activities, pharmacokinetic properties, and clinical efficacy for the treatment of chronic hepatitis C. Detailed physicochemical and biological characterization studies of PEG Intron revealed its composition of pegylated positional isomers and the specific anti-viral activity associated with each of them. Pegylation of Intron A at pH 6.5 results in a mixture of > or = 95% mono-pegylated isoforms with the predominant species (approximately 50%) derivatized to the His(34) residue with the remaining positional isomers pegylated at various lysines, the N-terminal cysteine, as well as serine, tyrosine, and another histidine residue. The anti-viral activity for each pegylated isomer showed that the highest specific activity (37%) was associated with the His(34)-pegylated isomer. Though pegylation decreases the specific activity of the interferon alpha-2b protein in vitro, the potency of PEG Intron was comparable to the Intron A standard at both the molecular and cellular level. The substituted IFN had an enhanced pharmacokinetic profile in both animal and human studies, and, when combined with ribavirin, was very effective in reducing hepatitis C viral load and maintaining sustained viral suppression in patients.