title: Correlations between in vitro potency of polyethylene glycol-protein conjugates and their chromatographic behavior.
abstract: Pegylation is the most widely used and accepted methodology for half-life extension of biopharmaceutical drugs that also improves physicochemical and biological characteristics of proteins considerably. Most of the positive pharmacological effects of pegylated proteins are believed to be related to an increased hydrodynamic volume and molecular size. To explore the size impact of polyethylene glycol (PEG) on in vitro potency, a series of well-defined conjugates of interferon alpha-2b (IFN) were prepared with PEGs of different lengths and shapes specifically attached to the N-terminal amino group of the protein. Specificity of the attachment was confirmed by peptide mapping and mass spectroscopy. When potency values determined by reporter gene assay were correlated with methods for molecular weight and size characterization, such as size exclusion chromatography and dynamic light scattering, rough parallels were found. Unexpectedly, the retention times on cation exchange chromatography showed much higher correlation with experimentally determined in vitro potency. It appears that in a series of N-terminally pegylated IFNs, their in vitro potency could be predicted from the retention times on the cation exchange chromatography columns, probably because both methods reflect not only the influence of molecular size but also the impact of protein masking exerted by attached PEG moiety.
authors.affiliation: National Institute of Chemistry, SI-1000 Ljubljana, Slovenia.
authors.forename: Simon
authors.initials: S
authors.lastname: Caserman
authors.forename: Mateja
authors.initials: M
authors.lastname: Kusterle
authors.forename: Menci
authors.initials: M
authors.lastname: Kunstelj
authors.forename: Tanja
authors.initials: T
authors.lastname: Milunović
authors.forename: Mark
authors.initials: M
authors.lastname: Schiefermeier
authors.forename: Simona
authors.initials: S
authors.lastname: Jevsevar
authors.forename: Vladka Gaberc
authors.initials: VG
authors.lastname: Porekar
mesh_terms: D002411:Cation Exchange Resins; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006001:Glycoconjugates / Q000737:chemistry*; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry*; D011994:Recombinant Proteins
chemical_list: D002411:Cation Exchange Resins; D006001:Glycoconjugates; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D011092:Polyethylene Glycols