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.

[1]  Eisen Hn.  Ultraviolet absorption spectroscopy of immune precipitates. , 1948, Journal of immunology.

[2]  R. Zinkernagel,et al.  T helper cell‐independent neutralizing B cell response against vesicular stomatitis virus: Role of antigen patterns in B cell induction? , 1995, European journal of immunology.

[3]  John F. Carpenter,et al.  Physical Stability of Proteins in Aqueous Solution: Mechanism and Driving Forces in Nonnative Protein Aggregation , 2003, Pharmaceutical Research.

[4]  A. Beldarraín,et al.  Purification and conformational properties of a human interferon α2b produced in Escherichia coli , 2001, Biotechnology and applied biochemistry.

[5]  Jochem Alsenz,et al.  Protein Aggregates Seem to Play a Key Role Among the Parameters Influencing the Antigenicity of Interferon Alpha (IFN-α) in Normal and Transgenic Mice , 1997, Pharmaceutical Research.

[6]  R. Zinkernagel,et al.  The influence of antigen organization on B cell responsiveness. , 1993, Science.

[7]  R. Borchardt,et al.  Stability of Protein Pharmaceuticals , 1989, Pharmaceutical Research.

[8]  S Pestka,et al.  Biological properties of recombinant alpha-interferons: 40th anniversary of the discovery of interferons. , 1998, Cancer research.

[9]  Huub Schellekens,et al.  Bioequivalence and the immunogenicity of biopharmaceuticals , 2002, Nature Reviews Drug Discovery.

[10]  D. Kalonia,et al.  Temperature- and pH-Induced Multiple Partially Unfolded States of Recombinant Human Interferon-α2a: Possible Implications in Protein Stability , 2003, Pharmaceutical Research.

[11]  J C Ryff,et al.  Clinical investigation of the immunogenicity of interferon-alpha 2a. , 1997, Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research.

[12]  Bhuwan Dhingra,et al.  A compendium and hydropathy/flexibility analysis of common reactive sites in proteins: reactivity at Asn, Asp, Gln, and Met motifs in neutral pH solution. , 1996, Pharmaceutical biotechnology.

[13]  V. Bumelis,et al.  Comparative effects of stabilizing additives on the rates of heat inactivation of recombinant human interferon alpha-2b in solution. , 2001, Antiviral research.

[14]  A. Whitty,et al.  Structural and functional differences between glycosylated and non-glycosylated forms of human interferon-beta (IFN-beta). , 1998, Pharmaceutical research.

[15]  J. Alsenz,et al.  Development and Use of Enzyme-Linked Immunosorbent Assays (ELISA) for the Detection of Protein Aggregates in Interferon-Alpha (IFN-α) Formulations , 1997, Pharmaceutical Research.

[16]  A. Savitzky,et al.  Smoothing and Differentiation of Data by Simplified Least Squares Procedures. , 1964 .

[17]  Adrian Whitty,et al.  Structural and Functional Differences Between Glycosylated and Non-glycosylated Forms of Human Interferon-β (IFN-β) , 1998, Pharmaceutical Research.

[18]  Heterologous boosting increases immunogenicity of chimeric papillomavirus virus-like particle vaccines. , 2003, Vaccine.

[19]  S. Pestka,et al.  Interferon immunogenicity: preclinical evaluation of interferon-alpha 2a. , 1997, Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research.

[20]  Huub Schellekens,et al.  Structure-Immunogenicity Relationships of Therapeutic Proteins , 2004, Pharmaceutical Research.

[21]  W V Moore,et al.  Role of aggregated human growth hormone (hGH) in development of antibodies to hGH. , 1980, The Journal of clinical endocrinology and metabolism.

[22]  Petra Lenz,et al.  Determinants of Autoantibody Induction by Conjugated Papillomavirus Virus-Like Particles , 2002, The Journal of Immunology.

[23]  E. Hochuli,et al.  Interferon immunogenicity: technical evaluation of interferon-alpha 2a. , 1997, Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research.

[24]  M. Slijper,et al.  Chapter 6 Structural characterization and immunogenicity in wildtype and immune tolerant mice of degraded recombinant human interferon alpha 2 b , 2005 .

[25]  D. Kalonia,et al.  Second derivative tryptophan fluorescence spectroscopy as a tool to characterize partially unfolded intermediates of proteins. , 2005, International journal of pharmaceutics.

[26]  R. Zinkernagel,et al.  Role of Repetitive Antigen Patterns for Induction of Antibodies Against Antibodies , 1997, The Journal of experimental medicine.

[27]  L. Ruiz,et al.  Long-term stabilization of recombinant human interferon alpha 2b in aqueous solution without serum albumin. , 2003, International journal of pharmaceutics.

[28]  G. Peterson,et al.  A simplification of the protein assay method of Lowry et al. which is more generally applicable. , 1977, Analytical biochemistry.

[29]  W Wang,et al.  Instability, stabilization, and formulation of liquid protein pharmaceuticals. , 1999, International journal of pharmaceutics.

[30]  G. Schernthaner,et al.  Immunogenicity and Allergenic Potential of Animal and Human Insulins , 1993, Diabetes Care.

[31]  D. Robbins,et al.  Antibodies to Covalent Aggregates of Insulin in Blood of Insulin-Using Diabetic Patients , 1987, Diabetes.

[32]  W. Jiskoot,et al.  Development of a Transgenic Mouse Model Immune Tolerant for Human Interferon Beta , 2005, Pharmaceutical Research.

[33]  D. Lowy,et al.  Conjugation of a self-antigen to papillomavirus-like particles allows for efficient induction of protective autoantibodies. , 2001, The Journal of clinical investigation.

[34]  J. Sadler,et al.  A Covalent Oxidoreductase Intermediate in Propeptide-dependent von Willebrand Factor Multimerization* , 2004, Journal of Biological Chemistry.

[35]  Ryff Jc,et al.  Clinical investigation of the immunogenicity of interferon-alpha 2a. , 1997 .