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Investigation of the Immunogenicity of Different Types of Aggregates of a Murine Monoclonal Antibody in Mice

Pharmaceutical Research volume 32, pages 430–444 (2015)Cite this article

Abstract

Purpose

The potential contribution of protein aggregates to the unwanted immunogenicity of protein pharmaceuticals is a major concern. In the present study a murine monoclonal antibody was utilized to study the immunogenicity of different types of aggregates in mice. Samples containing defined types of aggregates were prepared by processes such as stirring, agitation, exposure to ultraviolet (UV) light and exposure to elevated temperatures.

Methods

Aggregates were analyzed by size-exclusion chromatography, light obscuration, turbidimetry, infrared (IR) spectroscopy and UV spectroscopy. Samples were separated into fractions based on aggregate size by asymmetrical flow field-flow fractionation or by centrifugation. Samples containing different types and sizes of aggregates were subsequently administered to C57BL/6 J and BALB/c mice, and serum was analyzed for the presence of anti-IgG1, anti-IgG2a, anti-IgG2b and anti-IgG3 antibodies. In addition, the pharmacokinetic profile of the murine antibody was investigated.

Results

In this study, samples containing high numbers of different types of aggregates were administered in order to challenge the in vivo system. The magnitude of immune response depends on the nature of the aggregates. The most immunogenic aggregates were of relatively large and insoluble nature, with perturbed, non-native structures.

Conclusion

This study shows that not all protein drug aggregates are equally immunogenic.

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Abbreviations

ADAs:

Anti-drug antibodies

AF4:

Asymmetric flow field flow fractionation

ATR:

Attenuated total reflection

AUC:

Area under the curve

DMSO:

Dimethylsulfoxide

ELISA:

Enzyme-linked immunosorbent assay

FNU:

Formazine nephelometric units

HRP:

Horseradish peroxidase

IR:

Infrared spectroscopy

mAb1:

Monoclonal antibody

MALLS:

Multi angle laser light scattering

PBS:

Phosphate buffered saline

RI:

Refractive index

UV:

Ultraviolet light

References

  1. Berger M, Shankar V, Vafai A. Therapeutic applications of monoclonal antibodies. Am J Med Sci. 2002;324(1):14–30.

    Article  PubMed  Google Scholar 

  2. Koren E, Smith HW, Shores E, Shankar G, Finco-Kent D, Rup B, et al. Recommendations on risk-based strategies for detection and characterization of antibodies against biotechnology products. J Immunol Methods. 2008;333(1–2):1–9. Epub 2008/02/16.

    Article  CAS  PubMed  Google Scholar 

  3. Tamilvanan S, Raja NL, Sa B, Basu SK. Clinical concerns of immunogenicity produced at cellular levels by biopharmaceuticals following their parenteral administration into human body. J Drug Target. 2010;18(7):489–98.

    Article  CAS  PubMed  Google Scholar 

  4. Schellekens H. Immunogenicity of therapeutic proteins. Nephrol Dial Transplant. 2003;18(7):1257–9.

    Article  CAS  PubMed  Google Scholar 

  5. Singh SK. Impact of product-related factors on immunogenicity of biotherapeutics. J Pharm Sci. 2011;100(2):354–87.

    Article  CAS  PubMed  Google Scholar 

  6. Yanai H, Hanauer SB. Assessing response and loss of response to biological therapies in IBD. Am J Gastroenterol. 2011;106(4):685–98.

    Article  CAS  PubMed  Google Scholar 

  7. Swanson SJ, editor. Immunogenicity of Therapeutic Proteins. Hoboken: Wiley; 2010.

    Google Scholar 

  8. Shankar G, Pendley C, Stein KE. A risk-based bioanalytical strategy for the assessment of antibody immune responses against biological drugs. Nat Biotechnol. 2007;25(5):555–61. Epub 2007/05/08.

    Article  CAS  PubMed  Google Scholar 

  9. Schellekens H. Immunogenicity of protein therapeutics, or how to make antibodies without T-cells. Inflamm Res. 2007;56:S351–S2.

    Google Scholar 

  10. Schellekens H. Factors influencing the immunogenicity of therapeutic proteins. Nephrol Dial Transplant. 2005;20:3–9.

    Google Scholar 

  11. Schellekens H, Casadevall N. Immunogenicity of recombinant human proteins: causes and consequences. J Neurol. 2004;251:4–9.

    Article  Google Scholar 

  12. Schellekens H. The immunogenicity of biopharmaceuticals. Neurology. 2003;61(9):S11–S2.

    Article  PubMed  Google Scholar 

  13. Schellekens H. Relationship between biopharmaceutical immunogenicity of epoetin alfa and pure red cell aplasia. Curr Med Res Opin. 2003;19(5):433–4.

    Article  CAS  PubMed  Google Scholar 

  14. Schellekens H. Immunogenicity of therapeutic proteins: clinical implications and future prospects. Clin Ther. 2002;24(11):1720–40.

    Article  CAS  PubMed  Google Scholar 

  15. Rosenberg AS, Worobec A. A risk-based approach to immunogenicity concerns of therapeutic protein products part 1 considering consequences of the immune response to a protein. Biopharm Int. 2004;17(11):22−+.

    Google Scholar 

  16. Rosenberg A, editor. FDA Perspective on Immunogenicity Testing- A Risk Based Analysis. Bethesda, MD; 2003.

  17. Petersen B, Bendtzen K, Koch-Henriksen N, Ravnborg M, Ross C, Sorensen PS. Persistence of neutralizing antibodies after discontinuation of IFN beta therapy in patients with relapsing-remitting multiple sclerosis. Mult Scler. 2006;12(3):247–52.

    Article  PubMed  Google Scholar 

  18. De Groot AS, Scott DW. Immunogenicity of protein therapeutics. Trends Immunol. 2007;28(11):482–90.

    Article  PubMed  Google Scholar 

  19. Antonelli G, Dianzani F. Development of antibodies to interferon beta in patients: technical and biological aspects. Eur Cytokine Netw. 1999;10(3):413–22.

    CAS  PubMed  Google Scholar 

  20. Schellekens H. Immunologic mechanisms of EPO-associated pure red cell aplasia. Best Pract Res Clin Haematol. 2005;18(3):473–80.

    Article  CAS  PubMed  Google Scholar 

  21. Schernthaner G. Immunogenicity and allergenic potential of animal and human insulins. Diabetes Care. 1993;16 Suppl 3:155–65.

    Article  PubMed  Google Scholar 

  22. Goodin DS, Frohman EM, Hurwitz B, O’Connor PW, Oger JJ, Reder AT, et al. Neutralizing antibodies to interferon beta: assessment of their clinical and radiographic impact: an evidence report: report of the therapeutics and technology assessment subcommittee of the American academy of neurology. Neurology. 2007;68(13):977–84.

    Article  CAS  PubMed  Google Scholar 

  23. Ring J, Stephan W, Brendel W. Anaphylactoid reactions to infusions of plasma-protein and human-serum albumin - role of aggregated proteins and of stabilizers added during production. Clin Allergy. 1979;9(1):89–97.

    Article  CAS  PubMed  Google Scholar 

  24. Christian CL. Studies of aggregated γ-globulin: II effect in vivo. J Immunol. 1960;84(1):117–21.

    CAS  PubMed  Google Scholar 

  25. Roskos LK, Davis CG, Schwab GM. The clinical pharmacology of therapeutic monoclonal antibodies. Drug Dev Res. 2004;61(3):108–20.

    Article  CAS  Google Scholar 

  26. Pendley C, Schantz A, Wagner C. Immunogenicity of therapeutic monoclonal antibodies. Curr Opin Mol Ther. 2003;5(2):172–9.

    CAS  PubMed  Google Scholar 

  27. Hermeling S, Aranha L, Damen JMA, Slijper M, Schellekens H, Crommelin DJA, et al. Structural characterization and immunogenicity in wild-type and immune tolerant mice of degraded recombinant human interferon Alpha2b. Pharm Res. 2005;22(12):1997–2006.

    Article  CAS  PubMed  Google Scholar 

  28. Braun A, Kwee L, Labow MA, Alsenz J. Protein aggregates seem to play a key role among the parameters influencing the antigenicity of interferon alpha (IFN-alpha ) in normal and transgenic mice. Pharm Res. 1997;14(10):1472–8.

    Article  CAS  PubMed  Google Scholar 

  29. Van Beers MMC, Gilli F, Schellekens H, Randolph TW, Jiskoot W. Immunogenicity of recombinant human interferon beta interacting with particles of glass, metal, and polystyrene. J Pharm Sci. 2012;101(1):187–99.

    Article  PubMed  Google Scholar 

  30. Hesterberg LK, Seefeldt MB, Carpenter JF, Randolph TW. High-Hydrostatic pressure refolding of proteins. Genet Eng News. 2005;25(4):46–7.

    Google Scholar 

  31. Fradkin AH, Carpenter JF, Randolph TW. Immunogenicity of aggregates of recombinant human growth hormone in mouse models. J Pharm Sci. 2009;98(9):3247–64.

    Article  CAS  PubMed  Google Scholar 

  32. van Beers MMC, Sauerborn M, Gilli F, Brinks V, Schellekens H, Jiskoot W. Aggregated recombinant human interferon beta induces antibodies but no memory in immune-tolerant transgenic mice. Pharm Res. 2010;27(9):1812–24.

    Article  PubMed Central  PubMed  Google Scholar 

  33. Fradkin AH, Carpenter JF, Randolph TW. Glass particles as an adjuvant: a model for adverse immunogenicity of therapeutic proteins. J Pharm Sci. 2011;100(11):4953–64.

    Article  CAS  PubMed  Google Scholar 

  34. Brinks V, Jiskoot W, Schellekens H. Immunogenicity of therapeutic proteins: the use of animal models. Pharm Res. 2011;28(10):2379–85.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Hwang WYK, Foote J. Immunogenicity of engineered antibodies. Methods (San Diego, CA, U S). 2005;36(1):3–10.

    Article  CAS  Google Scholar 

  36. Schoeneich C. Light-induced oxidation and aggregation of proteins: potential immunogenicity consequences. Workshop on Protein Aggregation and Immunogenicity; July, 2010; Breckenridge, CO, July 20–22, 2010

  37. Wang W. Protein aggregation and its inhibition in biopharmaceutics. Int J Pharm. 2005;289(1–2):1–30.

    Article  CAS  PubMed  Google Scholar 

  38. Watanabe H, Numata K, Ito T, Takagi K, Matsukawa A. Innate immune response in Th1- and Th2-dominant mouse strains. Shock. 2004;22(5):460–6.

    Article  CAS  PubMed  Google Scholar 

  39. PhEur 2.2.1. Clarity and degree of opalescence of liquids. European Directorate for the Quality of Medicine (EDQM). 7th edition; 2011.

  40. PhEur 0169. Monograph “Water for injections”. European Directorate for the Quality of Medicine (EDQM). 7th edition; 2011.

  41. Dintzis HM, Dintzis RZ, Vogelstein B. Molecular determinants of immunogenicity: the immunon model of immune response. Proc Natl Acad Sci U S A. 1976;73(10):3671–5.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  42. Martin RM, Brady JL, Lew AM. The need for IgG2c specific antiserum when isotyping antibodies from C57BL/6 and NOD mice. J Immunol Methods. 1998;212(2):187–92.

    Article  CAS  PubMed  Google Scholar 

  43. Petrovsky N, Aguilar JC. Vaccine adjuvants: current state and future trends. Immunol Cell Biol. 2004;82(5):488–96.

    Article  CAS  PubMed  Google Scholar 

  44. Freitag AJ, Wittmann K, Winter G, Myschik J. The preparative use of flow field-flow fractionation. LCGC Europe. 2011;24(3):134.

    CAS  Google Scholar 

  45. Shomali M, Freitag A, Engert J, Siedler M, Kaymakcalan Z, Winter G, et al. Antibody responses in mice to particles formed from adsorption of a murine monoclonal antibody onto glass microparticles. J Pharm Sci. 2014;103(1):78–89.

    Article  CAS  PubMed  Google Scholar 

  46. Mills CD, Kincaid K, Alt JM, Heilman MJ, Hill AM. M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol. 2000;164(12):6166–73.

    Article  CAS  PubMed  Google Scholar 

  47. Coutelier JP, Van der Logt JTM, Heessen FWA, Vink A, Van Snick A. Virally induced modulation of murine IgG antibody subclasses. J Exp Med. 1988;168(6):2373–8.

    Article  CAS  PubMed  Google Scholar 

  48. Stevens TL, Bossie A, Sanders VM, Fernandez-Botran R, Coffman RL, Mosmann TR, et al. Regulation of antibody isotype secretion by subsets of antigen-specific helper T cells. Nature (London). 1988;334(6179):255–8.

    Article  CAS  Google Scholar 

  49. Ramakrishna C, Ravi V, Desai A, Subbakrishna DK, Shankar SK, Chandramuki A. T helper responses to japanese encephalitis virus infection are dependent on the route of inoculation and the strain of mouse used. J Gen Virol. 2003;84(6):1559–67.

    Article  CAS  PubMed  Google Scholar 

  50. Feltquate DM, Heaney S, Webster RG, Robinson HL. Different T helper cell types and antibody isotypes generated by saline and gene gun DNA immunization. J Immunol. 1997;158(5):2278–84.

    CAS  PubMed  Google Scholar 

  51. Hermeling S, Schellekens H, Maas C, Gebbink MFBG, Crommelin DJA, Jiskoot W. Antibody response to aggregated human interferon alpha2b in wild-type and transgenic immune tolerant mice depends on type and level of aggregation. J Pharm Sci. 2006;95(5):1084–96.

    Article  CAS  PubMed  Google Scholar 

  52. Vollmar D. Immunologie - Grundlagen und Wirkstoffe. 1st ed. München, Frankfurt am Main: Wissenschaftliche Verlagsgesellschaft mbH Stuttgart; 2005.

    Google Scholar 

  53. Haley PJ. Species differences in the structure and function of the immune system. Toxicology. 2003;188(1):49–71.

    Article  CAS  PubMed  Google Scholar 

  54. Filipe V, Que I, Carpenter J, Löwik C, Jiskoot W. In vivo fluorescence imaging of IgG1 aggregates after subcutaneous and intravenous injection in mice. Pharm Res. 2014;31(1):216–27.

    Article  CAS  PubMed  Google Scholar 

  55. Kijanka G, Prokopowicz M, Schellekens H, Brinks V. Influence of aggregation and route of injection on the biodistribution of mouse serum albumin. PLoS One. 2014;9(1):1–9.

    Article  Google Scholar 

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Acknowledgments and Disclosures

The authors would like to thank AbbVie Inc. for providing the protein and financial support.

Disclosure of Potential Conflicts of Interest

Zehra Kaymakcalan and Michael Siedler are employees of AbbVie and are Abbvie stockholders.

The University of Colorado and the Ludwig-Maximilians-University Munich received research funds from AbbVie Inc. (former Abbott Laboratories) to conduct the study.

AbbVie (former Abbott Laboratories) provided financial support, provided the murine antibody used in this study, as well as resources to support the in-vivo studies and the bioanalytical characterization.

Furthermore, AbbVie authors were involved in study design, research, analysis, data collection, interpretation of data, reviewing and approving the publication.

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Authors and Affiliations

  1. Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximilians-University Munich, Butenandtstr. 5, D-81377, Munich, Germany

    Angelika J. Freitag, Gerhard Winter & Julia Engert

  2. Department of Chemical and Biological Engineering, Center for Pharmaceutical Biotechnology, University of Colorado, Boulder, Colorado, 80309, USA

    Maliheh Shomali & Theodore W. Randolph

  3. AbbVie Bioresearch Center, 100 Research Drive, Worcester, Massachusetts, 01605, USA

    Michael Siedler & Zehra Kaymakcalan

  4. Department of Pharmaceutical Sciences, Center for Pharmaceutical Biotechnology, University of Colorado Health Sciences Center, Box 238, Denver, Colorado, 80262, USA

    John F. Carpenter

  5. Center for Integrated Protein Science Munich CiPSM and Department of Pharmacy – Center for Drug Research, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, D-81377, Munich, Germany

    Stylianos Michalakis & Martin Biel

Authors
  1. Angelika J. Freitag
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  2. Maliheh Shomali
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  3. Stylianos Michalakis
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  4. Martin Biel
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  6. Zehra Kaymakcalan
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  7. John F. Carpenter
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  8. Theodore W. Randolph
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  9. Gerhard Winter
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  10. Julia Engert
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Corresponding author

Correspondence to Julia Engert.

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Freitag, A.J., Shomali, M., Michalakis, S. et al. Investigation of the Immunogenicity of Different Types of Aggregates of a Murine Monoclonal Antibody in Mice. Pharm Res 32, 430–444 (2015). https://doi.org/10.1007/s11095-014-1472-6

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  • DOI: https://doi.org/10.1007/s11095-014-1472-6

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