Antibody response to aggregated human interferon alpha2b in wild-type and transgenic immune tolerant mice depends on type and level of aggregation.
The aim of this study was to determine the sensitivity of transgenic immune tolerant mice for the type and level of aggregation of recombinant human interferon alpha2b (rhIFNalpha2b). RhIFNalpha2b was aggregated by metal-catalyzed oxidation or by incubation at elevated temperature and various pHs. Native rhIFNalpha2b was mixed with oxidized rhIFNalpha2b at different ratios to obtain samples with different aggregation levels. The preparations were characterized by UV and fluorescence spectroscopy, gel permeation chromatography (GPC), dynamic light scattering (DLS), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, and ELISA. The immunogenicity was evaluated in wild-type mice and transgenic mice immune tolerant for hIFNalpha2. Sera were analyzed by ELISA for the presence of rhIFNalpha2b-specific antibodies. The oxidized and aged preparations widely differed regarding the level and nature of aggregates. All preparations containing aggregates increased the immune response in the wild-type mice as compared to native rhIFNalpha2b and were able to break the tolerance of the transgenic mice. The more native-like the conformation of the aggregated proteins, the more immunogenic the preparations were in the transgenic mice. The native-like aggregates prepared via metal catalysis induced a dose-dependent loss of tolerance in the transgenic mice. In conclusion, the transgenic mouse model can be used to screen rhIFNalpha2b formulations for low levels of immunogenic aggregates obtained under accelerated storage conditions.
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands.
Suzanne
S
Hermeling
Huub
H
Schellekens
Coen
C
Maas
Martijn F B G
MF
Gebbink
Daan J A
DJ
Crommelin
Wim
W
Jiskoot
D000818:Animals; D000917:Antibody Formation / Q000187:drug effects; D015153:Blotting, Western; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007108:Immune Tolerance / Q000276:immunology*; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry / Q000276:immunology*; D008027:Light; D051379:Mice; D008822:Mice, Transgenic; D011994:Recombinant Proteins; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000939:Epitopes; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins