Pharmaceutical Research

, Volume 22, Issue 12, pp 1997–2006 | Cite as

Structural Characterization and Immunogenicity in Wild-Type and Immune Tolerant Mice of Degraded Recombinant Human Interferon Alpha2b

  • Suzanne Hermeling
  • Liliana Aranha
  • J. Mirjam A. Damen
  • Monique Slijper
  • Huub Schellekens
  • Daan J. A. Crommelin
  • Wim Jiskoot
Research Paper


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 (rhIFNα2b).


RhIFNα2b 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 hIFNα2. Serum antibodies were detected by enzyme-linked immunosorbent assay or surface plasmon resonance.


M-rhIFNα2b contained covalently aggregated rhIFNα2b with three methionines partly oxidized to methionine sulfoxides. G-rhIFNα2b contained covalent aggregates and did not show changes in secondary structure. H-rhIFNα2b was only chemically changed with four partly oxidized methionines. B-rhIFNα2b was largely unfolded and heavily aggregated. Nontreated (N) rhIFNα2b was immunogenic in the wild-type mice but not in the transgenic mice, showing that the latter were immune tolerant for rhIFNα2b. The anti-rhIFNα2b antibody levels in the wild-type mice depended on the degradation product: M-rhIFNα2b > H-rhIFNα2b ∼ N-rhIFNα2b ≫ B-rhIFNα2b; G-rhIFNα2b did not induce anti-rhIFNα2b antibodies. In the transgenic mice, only M-rhIFNα2b could break the immune tolerance.


RhIFNα2b immunogenicity is related to its structural integrity. Moreover, the immunogenicity of aggregated rhIFNα2b depends on the structure and orientation of the constituent protein molecules and/or on the aggregate size.

Key Words

aggregates immune tolerance immunogenicity interferon alpha2 protein structure transgenic mice 



2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)


bovine serum albumin


circular dichroism


dynamic light scattering




enzyme-linked immunosorbent assay


electrospray ionization-time of flight


gel permeation chromatography


human interferon alpha2




matrix-assisted laser desorption ionization time of flight/time of flight


polyacrylamide gel electrophoresis


sodium phosphate buffer, pH 7.2


phosphate-buffered saline


recombinant human interferon alpha2b


reverse-phase high-pressure liquid chromatography




sodium dodecyl sulfate


surface plasmon resonance


trifluoroacetic acid



The authors wish to thank Dr. Viscomi and Lucia Scapol for supplying the rhIFNα2b solutions, the standard anti-rhIFNα2b serum, and for valuable discussions. Dr. Pestka is kindly thanked for supplying the transgenic immune tolerant mice. Janny Westdijk is kindly acknowledged for her help with the Biacore experiments and valuable discussions. We thank Ronald van Ooijen and Georgina Gal for performing the mass spectrometric analyses. This work was financially supported by the European Union through the 5th Framework Program “Competitive and Sustainable Growth,” LYOPRO project (Contract no. G1RD-CT2002-00736).


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Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • Suzanne Hermeling
    • 1
    • 2
  • Liliana Aranha
    • 2
  • J. Mirjam A. Damen
    • 3
  • Monique Slijper
    • 3
  • Huub Schellekens
    • 1
  • Daan J. A. Crommelin
    • 2
  • Wim Jiskoot
    • 2
  1. 1.Central Laboratory Animal InstituteUtrecht UniversityUtrechtThe Netherlands
  2. 2.Department of Pharmaceutics, Faculty of Pharmaceutical SciencesUtrecht Institute for Pharmaceutical Sciences (UIPS)UtrechtThe Netherlands
  3. 3.Department of Biomolecular Mass SpectrometryUtrecht Institute for Pharmaceutical Sciences (UIPS) and Bijvoet Center for Biomolecular ResearchUtrechtThe Netherlands

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