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The protein protocols handbook

The protein protocols handbook. In contrast with wild-type mice, transgenic mice did not show NABs, nor did they respond to the rechallenge. Conclusions The immunogenicity of the products in transgenic mice, unlike in wild-type mice, varied. In the LDN193189 transgenic mice, neither NABs nor immunological memory developed. The immunogenicity of rhIFN in a model reflecting the human immune system depends on the presence and the characteristics of aggregates. test, two-tailed) were performed between groups with 100% responders on log10 converted titers. Asterisks indicate that titers are significantly ( em p /em ? ?0.04) higher after the rechallenge than before. These results comply with the lack of antibody response observed in patients who, after a wash-out period, switched to Avonex?-rhIFN-1a treatment after having developed high levels of anti-rhIFN-1b antibodies following Betaferon? treatment (15). Despite the cross-reactivity of anti-rhIFN antibodies, levels of pre-occurring BABs or NABs in patients did not increase after switching the treatment from Betaferon? to Avonex? (15,40), from rhIFN-1a to high-dose intravenous rhIFN-1b (41), and from 1.6 to 8 8 million international units of rhIFN-1b (42), without a wash-out period. Especially patients with low titers may even reconvert to antibody negativity while treatment continues, independent of the type of rhIFN that is administered (40,43C46). The observed lack of immunological memory in immune-tolerant mice as well as in RR-MS patients may be characteristic for the breakage of B-cell tolerance for recombinant human therapeutic proteins. FINAL REMARKS AND CONCLUSIONS Bulk rhIFN-1a, which contained mainly non-covalently bound aggregates, induced a transient immune response in approximately 40% of the transgenic mice. Filtration of the bulk product reduced the aggregation level, and reformulation in another buffer prevented the formation of new aggregates, thereby completely abolishing its potency to break immune tolerance. Despite the high percentage of aggregates in stressed rhIFN-1a, only about 30% of the transgenic mice receiving this product showed antibodies against rhIFN-1a. This is possibly explained by the absence of native epitopes in the covalent non-reducible aggregates as shown by Western blotting. Preservation of the native structure of the protein is a prerequisite for aggregates to break the tolerance of transgenic, immune-tolerant mice (8). In addition to BABs, the wild-type mice formed NABs and immunological memory for the protein after 3-week administration of any of the rhIFN-1a samples or Betaferon?. This study confirms that wild-type animals cannot be used to study the immunogenicity of human therapeutic proteins, and immune-tolerant animal models are needed (47). In this paper, transgenic mouse models showed that protein aggregates are able to break the immune tolerance for rhIFN. The potency of LDN193189 the aggregates to break tolerance not only depends on aggregate percentage but also largely on their physical properties such as degree of denaturation, molecular orientation and size. Moreover, we demonstrated that the breaking of immune tolerance for rhIFN in transgenic mice is characterized by the absence of NABs and immunological memory and thereby differs substantially from a classical T-cell-dependent immune response. ACKNOWLEDGEMENTS This research was financially supported by the European Community under its 6th Framework (project NABINMS, contract number 018926). Biogen Idec Inc. is acknowledged for kindly providing test products. We thank Susan Goelz for her valuable suggestions and discussions. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. ABBREVIATIONS BABsbinding antibodiesECDequivalent circular diameterhIFNhuman interferon HSAhuman serum albuminIgGimmunoglobulin Gi.p.intraperitoneallyMxAmyxovirus resistant protein ANABsneutralizing antibodiesPDIpolydispersity indexrhIFNrecombinant GLUR3 human interferon betarhIFN-1arecombinant human interferon beta-1arhIFN-1brecombinant human interferon beta-1bRR-MSrelapsing-remitting multiple sclerosisSDSsodium dodecyl sulfateTRU/mlten-fold reduction units per ml REFERENCES 1. Schellekens H. Bioequivalence and the immunogenicity of biopharmaceuticals. Nat Rev Drug Discov. 2002;1:457C462. doi:?10.1038/nrd818. [PubMed] [CrossRef] [Google Scholar] 2. Antonelli G. Reflections on the immunogenicity of therapeutic proteins. 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