title: Physicochemical and biological assays for quality control of biopharmaceuticals: interferon alpha-2 case study.
abstract: A selection of physicochemical and biological assays were investigated for their utility in detecting changes in preparations of Interferon alpha-2a and Interferon alpha-2b (IFN-alpha 2a, IFN-alpha 2b), which had been subjected to stressed conditions, in order to create models of biopharmaceutical products containing product-related impurities. The stress treatments, which included oxidation of methionine residues and storage at elevated temperatures for different periods of time, were designed to induce various degrees of degradation, aggregation or oxidation of the interferon. Biological activity of the stressed preparations was assessed in three different in vitro cell-based bioassay systems: a late-stage anti-proliferative assay and early-stage assays measuring reporter gene activation or endogenous gene expression by quantitative real time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Relevant physicochemical methods such as SDS-PAGE, reverse phase (RP) chromatography, size-exclusion chromatography (SEC) and dynamic light scattering (DLS), proved their complementarity in detecting structural changes in the stressed preparations which were reflected by reductions in biological activity.
authors.affiliation: National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK. msilva@nibsc.ac.uk
authors.forename: Marta M C G
authors.initials: MM
authors.lastname: Silva
authors.forename: Baptiste
authors.initials: B
authors.lastname: Lamarre
authors.forename: Eleonora
authors.initials: E
authors.lastname: Cerasoli
authors.forename: Paulina
authors.initials: P
authors.lastname: Rakowska
authors.forename: Anna
authors.initials: A
authors.lastname: Hills
authors.forename: Marc J A
authors.initials: MJ
authors.lastname: Bailey
authors.forename: Jun X
authors.initials: JX
authors.lastname: Wheeler
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Burns
authors.forename: Rose E
authors.initials: RE
authors.lastname: Gaines-Das
authors.forename: Chris
authors.initials: C
authors.lastname: Jones
authors.forename: C Jane
authors.initials: CJ
authors.lastname: Robinson
mesh_terms: D001681:Biological Assay / Q000379:methods*; D001688:Biological Products / Q000737:chemistry* / Q000378:metabolism; D049109:Cell Proliferation; D002845:Chromatography / Q000379:methods; D015195:Drug Design; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000032:analysis* / Q000378:metabolism; D008715:Methionine / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013048:Specimen Handling; D013696:Temperature
chemical_list: D001688:Biological Products; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D008715:Methionine; D010100:Oxygen