title: Structural characterization and immunogenicity in wild-type and immune tolerant mice of degraded recombinant human interferon alpha2b.
abstract: OBJECTIVE
  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 (rhIFNalpha2b).
  
  
  METHODS
  RhIFNalpha2b 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 hIFNalpha2. Serum antibodies were detected by enzyme-linked immunosorbent assay or surface plasmon resonance.
  
  
  RESULTS
  M-rhIFNalpha2b contained covalently aggregated rhIFNalpha2b with three methionines partly oxidized to methionine sulfoxides. G-rhIFNalpha2b contained covalent aggregates and did not show changes in secondary structure. H-rhIFNalpha2b was only chemically changed with four partly oxidized methionines. B-rhIFNalpha2b was largely unfolded and heavily aggregated. Nontreated (N) rhIFNalpha2b was immunogenic in the wild-type mice but not in the transgenic mice, showing that the latter were immune tolerant for rhIFNalpha2b. The anti-rhIFNalpha2b antibody levels in the wild-type mice depended on the degradation product: M-rhIFNalpha2b > H-rhIFNalpha2b approximately N-rhIFNalpha2b >> B-rhIFNalpha2b; G-rhIFNalpha2b did not induce anti-rhIFNalpha2b antibodies. In the transgenic mice, only M-rhIFNalpha2b could break the immune tolerance.
  
  
  CONCLUSIONS
  RhIFNalpha2b immunogenicity is related to its structural integrity. Moreover, the immunogenicity of aggregated rhIFNalpha2b depends on the structure and orientation of the constituent protein molecules and/or on the aggregate size.
authors.affiliation: Central Laboratory Animal Institute, Utrecht University, Utrecht, The Netherlands. S.Hermeling@xs4all.nl
authors.forename: Suzanne
authors.initials: S
authors.lastname: Hermeling
authors.forename: Liliana
authors.initials: L
authors.lastname: Aranha
authors.forename: J Mirjam A
authors.initials: JM
authors.lastname: Damen
authors.forename: Monique
authors.initials: M
authors.lastname: Slijper
authors.forename: Huub
authors.initials: H
authors.lastname: Schellekens
authors.forename: Daan J A
authors.initials: DJ
authors.lastname: Crommelin
authors.forename: Wim
authors.initials: W
authors.lastname: Jiskoot
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005976:Glutaral / Q000737:chemistry; D006801:Humans; D007108:Immune Tolerance / Q000276:immunology*; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry* / Q000276:immunology*; D008027:Light; D013058:Mass Spectrometry; D008670:Metals; D051379:Mice; D008822:Mice, Transgenic; D010084:Oxidation-Reduction; D011994:Recombinant Proteins; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D020349:Surface Plasmon Resonance
chemical_list: D000077190:Interferon alpha-2; D016898:Interferon-alpha; D008670:Metals; D011994:Recombinant Proteins; D005976:Glutaral