CMC and regulatory considerations for polyethylene glycol-modified protein therapeutics

Abstract

The hepatitis C virus (HCV) is currently estimated to infect over 270 million people worldwide, with up to 4 million people newly infected each year. It is the leading cause of chronic liver disease and the most common reason for liver transplantation in the USA and Europe. The pegylated interferons (peginterferon alfa-2b: PEG-INTRON® and peginterferon alfa-2a: PEGASYS®), alone or in combination with ribavirin, are the current standard of care for patients with HCV. There are significant chemistry and manufacturing control (CMC) challenges in the manufacture and characterization of the pegylated interferons since these therapeutic drugs are composed of heterogeneous mixtures of mono-pegylated proteins with differing sites of attachment of polyethylene glycol (PEG) to the core interferon molecule, termed “positional isomers”. These CMC issues were comprehensively addressed during the development of PEG-INTRON®. Extensive characterization of the pegylated interferon in the drug substance and drug product, and the establishment of appropriate manufacturing controls, served as the foundation for a successful CMC strategy to obtain regulatory approval. This study describes the analytical strategies for quantitating the positional isomer populations which comprise PEG-INTRON®. Individual positional isomers were also isolated and characterized with respect to site of pegylation and in vitro biological potency. It was determined that different pegylation chemistries result in different mixtures of positional isomers. PEG-INTRON® is predominantly pegylated at histidine 34, demonstrated to be the most biologically active positional isomer of the pegylated interferons. In contrast, the antiviral activities of positional isomers generated by attachment of PEG to lysine residues were significantly lower. Thus, the differences in pegylation chemistry between PEG-INTRON® and PEGASYS® result in different distributions of pegylated positional isomers and differential biological activity profiles in vitro.