Oxidized and Aggregated Recombinant Human Interferon Beta is Immunogenic in Human Interferon Beta Transgenic Mice

To study the effect of oxidation on the structure of recombinant human interferon beta-1a (rhIFN beta-1a) and its immunogenicity in wild-type and immune-tolerant transgenic mice. Untreated rhIFN beta-1a was degraded by metal-catalyzed oxidation, H2O2-mediated oxidation, and guanidine-mediated unfolding/refolding. Four rhIFN beta-1a preparations with different levels of oxidation and aggregation were injected intraperitoneally in mice 15x during 3 weeks. Both binding and neutralizing antibodies were measured. All rhIFN beta-1a preparations contained substantial amounts of aggregates. Metal-catalyzed oxidized rhIFN beta-1a contained high levels of covalent aggregates as compared with untreated rhIFN beta-1a. H2O2-treated rhIFN beta-1a showed an increase in oligomer and unrecovered protein content by HP-SEC; RP-HPLC revealed protein oxidation. Guanidine-treated rhIFN beta-1a mostly consisted of dimers and oligomers and some non-covalent aggregates smaller in size than those in untreated rhIFN beta-1a. All degraded samples showed alterations in tertiary protein structure. Wild-type mice showed equally high antibody responses against all preparations. Transgenic mice were discriminative, showing elevated antibody responses against both metal-catalyzed oxidized and H2O2-treated rhIFN beta-1a as compared to untreated and guanidine-treated rhIFN beta-1a. Oxidation-mediated aggregation increased the immunogenicity of rhIFN beta-1a in transgenic mice, whereas aggregated preparations devoid of measurable oxidation levels were hardly immunogenic.