title: Oxidized and aggregated recombinant human interferon beta is immunogenic in human interferon beta transgenic mice.
abstract: OBJECTIVE
  To study the effect of oxidation on the structure of recombinant human interferon beta-1a (rhIFNβ-1a) and its immunogenicity in wild-type and immune-tolerant transgenic mice.
  
  
  METHODS
  Untreated rhIFNβ-1a was degraded by metal-catalyzed oxidation, H(2)O(2)-mediated oxidation, and guanidine-mediated unfolding/refolding. Four rhIFNβ-1a preparations with different levels of oxidation and aggregation were injected intraperitoneally in mice 15× during 3 weeks. Both binding and neutralizing antibodies were measured.
  
  
  RESULTS
  All rhIFNβ-1a preparations contained substantial amounts of aggregates. Metal-catalyzed oxidized rhIFNβ-1a contained high levels of covalent aggregates as compared with untreated rhIFNβ-1a. H(2)O(2)-treated rhIFNβ-1a showed an increase in oligomer and unrecovered protein content by HP-SEC; RP-HPLC revealed protein oxidation. Guanidine-treated rhIFNβ-1a mostly consisted of dimers and oligomers and some non-covalent aggregates smaller in size than those in untreated rhIFNβ-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 H(2)O(2)-treated rhIFNβ-1a as compared to untreated and guanidine-treated rhIFNβ-1a.
  
  
  CONCLUSIONS
  Oxidation-mediated aggregation increased the immunogenicity of rhIFNβ-1a in transgenic mice, whereas aggregated preparations devoid of measurable oxidation levels were hardly immunogenic.
authors.affiliation: Division of Drug Delivery Technology, Leiden/Amsterdam Center for Drug Research (LACDR), Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
authors.forename: Miranda M C
authors.initials: MM
authors.lastname: van Beers
authors.forename: Melody
authors.initials: M
authors.lastname: Sauerborn
authors.forename: Francesca
authors.initials: F
authors.lastname: Gilli
authors.forename: Vera
authors.initials: V
authors.lastname: Brinks
authors.forename: Huub
authors.initials: H
authors.lastname: Schellekens
authors.forename: Wim
authors.initials: W
authors.lastname: Jiskoot
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology; D000917:Antibody Formation / Q000276:immunology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D007108:Immune Tolerance / Q000276:immunology; D000068556:Interferon beta-1a; D016899:Interferon-beta / Q000737:chemistry* / Q000276:immunology* / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000276:immunology; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology
chemical_list: D057134:Antibodies, Neutralizing; D011994:Recombinant Proteins; D016899:Interferon-beta; D006861:Hydrogen Peroxide; D019791:Guanidine; D000068556:Interferon beta-1a