title: Immunogenicity of Recombinant Human Interferon Beta-1b in Immune-Tolerant Transgenic Mice Corresponds with the Biophysical Characteristics of Aggregates.
abstract: Determining to what extent biophysical characteristics of aggregates affect immunogenicity of therapeutic interferon beta-1b. Three recombinant human interferon beta-1b (rhIFNβ-1b) samples with different levels of aggregates generated by copper oxidation, thermal stress, or left untreated, as well as Avonex(®) drug substance and Betaferon(®) drug product, were injected intraperitoneally in nontransgenic and interferon beta transgenic FVB/N mice 5 times per week for 3 weeks. Antibodies against interferon beta were measured using enzyme-linked immunosorbent assay. UV and fluorescence spectroscopy, dynamic light scattering, size exclusion chromatography, reversed-phase high-performance liquid chromatography (RP-HPLC), fluid imaging microscopy, and resonant mass measurement, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, were used to characterize and quantitate aggregates in the 3 rhIFNβ preparations, to correlate biophysical characteristics with immunogenicity. In immune-tolerant interferon beta transgenic FVB/N mice, Betaferon drug product showed the highest immunogenicity, while Avonex drug substance showed the lowest level of immunogenicity. Of the 3 forms of rhIFNβ-1b, copper-oxidized rhIFNβ-1b showed lower immunogenicity than thermally stressed rhIFNβ-1b, despite containing larger aggregates. Both copper-oxidized rhIFNβ-1b and thermally stressed rhIFNβ-1b exhibited changes in protein structure as shown using fluorescence spectroscopy and RP-HPLC. Nontransgenic, nonimmune-tolerant FVB/N mice generated high antibody titers against all interferon beta samples tested. The level of immunogenicity and the breaking of tolerance in FVB/N transgenic mice are not only related to the level of aggregation but also depend on the size and structure of the aggregates.
authors.affiliation: 1 Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University , Utrecht, The Netherlands .
authors.forename: Mohadeseh Haji
authors.initials: MH
authors.lastname: Abdolvahab
authors.affiliation: 2 Department of Research and Development, Zistdaru Danesh Co. Ltd. , Tehran, Iran .
authors.forename: Ahmad
authors.initials: A
authors.lastname: Fazeli
authors.affiliation: 1 Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University , Utrecht, The Netherlands .
authors.forename: Andhyk
authors.initials: A
authors.lastname: Halim
authors.affiliation: 3 Division of Drug Delivery Technology, Gorlaeus Laboratories, Leiden Centre for Drug Research (LACDR), Leiden University , Leiden, The Netherlands .
authors.forename: Ahmad S
authors.initials: AS
authors.lastname: Sediq
authors.affiliation: 4 Department of Drug and Food Control, Faculty of Pharmacy and Pharmaceutical Quality Assurance Research Centre, Tehran University of Medical Sciences , Tehran, Iran .
authors.forename: Mohammad Reza
authors.initials: MR
authors.lastname: Fazeli
authors.affiliation: 1 Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University , Utrecht, The Netherlands .
authors.forename: Huub
authors.initials: H
authors.lastname: Schellekens
mesh_terms: D000818:Animals; D000906:Antibodies / Q000097:blood; D006801:Humans; D007108:Immune Tolerance; D007167:Immunotherapy / Q000379:methods*; D007274:Injections, Intraperitoneal; D000068556:Interferon beta-1a / Q000737:chemistry / Q000276:immunology*; D000068576:Interferon beta-1b / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D008822:Mice, Transgenic; D010084:Oxidation-Reduction; D066329:Protein Aggregates; D013696:Temperature
chemical_list: D000906:Antibodies; D066329:Protein Aggregates; D000068576:Interferon beta-1b; D000068556:Interferon beta-1a