Structural characterization and immunogenicity in wild-type and immune tolerant mice of degraded recombinant human interferon alpha2b

Purpose. This study was conducted to study the influence of protein structure on the immunogenicity in wild-type and immune tolerant mice of well-characterized degradation products of recombinant human interferon alpha2b (rhIFN alpha 2b). Methods. RhIFN alpha 2b was degraded by metal-catalyzed oxidation (M), cross-linking with glutaraldehyde (G), oxidation with hydrogen peroxide (H), and incubation in a boiling water bath (B). The products were characterized with UV absorption, circular dichroism and fluorescence spectroscopy, gel permeation chromatography, reverse-phase high-pressure liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and mass spectrometry. The immunogenicity of the products was evaluated in wild-type mice and in transgenic mice immune tolerant for hIFN alpha 2. Serum antibodies were detected by enzyme-linked immunosorbent assay or surface plasmon resonance. Results. M-rhIFN alpha 2b contained covalently aggregated rhIFN alpha 2b with three methionines partly oxidized to methionine sulfoxides. G-rhIFN alpha 2b contained covalent aggregates and did not show changes in secondary structure. H-rhIFN alpha 2b was only chemically changed with four partly oxidized methionines. B-rhIFN alpha 2b was largely unfolded and heavily aggregated. Nontreated (N) rhIFN alpha 2b was immunogenic in the wild-type mice but not in the transgenic mice, showing that the latter were immune tolerant for rhIFN alpha 2b. The anti-rhIFN alpha 2b antibody levels in the wild-type mice depended on the degradation product: M-rhIFN alpha 2b > H-rhIFN alpha 2b similar to N-rhIFN alpha 2b >> B-rhIFN alpha 2b; G-rhIFN alpha 2b did not induce anti-rhIFN alpha 2b antibodies. In the transgenic mice, only M-rhIFN alpha 2b could break the immune tolerance. Conclusions. RhIFN alpha 2b immunogenicity is related to its structural integrity. Moreover, the immunogenicity of aggregated rhIFN alpha 2b depends on the structure and orientation of the constituent protein molecules and/or on the aggregate size.