Advertisement

Pharmaceutical Research

, Volume 32, Issue 2, pp 430–444 | Cite as

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

  • Angelika J. Freitag
  • Maliheh Shomali
  • Stylianos Michalakis
  • Martin Biel
  • Michael Siedler
  • Zehra Kaymakcalan
  • John F. Carpenter
  • Theodore W. Randolph
  • Gerhard Winter
  • Julia EngertEmail author
Research Paper

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.

Key Words

Immunogenicity Monoclonal antibody Protein aggregates Protein particles Wild-type mice 

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

Notes

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.

References

  1. 1.
    Berger M, Shankar V, Vafai A. Therapeutic applications of monoclonal antibodies. Am J Med Sci. 2002;324(1):14–30.PubMedCrossRefGoogle Scholar
  2. 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.PubMedCrossRefGoogle Scholar
  3. 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.PubMedCrossRefGoogle Scholar
  4. 4.
    Schellekens H. Immunogenicity of therapeutic proteins. Nephrol Dial Transplant. 2003;18(7):1257–9.PubMedCrossRefGoogle Scholar
  5. 5.
    Singh SK. Impact of product-related factors on immunogenicity of biotherapeutics. J Pharm Sci. 2011;100(2):354–87.PubMedCrossRefGoogle Scholar
  6. 6.
    Yanai H, Hanauer SB. Assessing response and loss of response to biological therapies in IBD. Am J Gastroenterol. 2011;106(4):685–98.PubMedCrossRefGoogle Scholar
  7. 7.
    Swanson SJ, editor. Immunogenicity of Therapeutic Proteins. Hoboken: Wiley; 2010.Google Scholar
  8. 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.PubMedCrossRefGoogle Scholar
  9. 9.
    Schellekens H. Immunogenicity of protein therapeutics, or how to make antibodies without T-cells. Inflamm Res. 2007;56:S351–S2.Google Scholar
  10. 10.
    Schellekens H. Factors influencing the immunogenicity of therapeutic proteins. Nephrol Dial Transplant. 2005;20:3–9.Google Scholar
  11. 11.
    Schellekens H, Casadevall N. Immunogenicity of recombinant human proteins: causes and consequences. J Neurol. 2004;251:4–9.CrossRefGoogle Scholar
  12. 12.
    Schellekens H. The immunogenicity of biopharmaceuticals. Neurology. 2003;61(9):S11–S2.PubMedCrossRefGoogle Scholar
  13. 13.
    Schellekens H. Relationship between biopharmaceutical immunogenicity of epoetin alfa and pure red cell aplasia. Curr Med Res Opin. 2003;19(5):433–4.PubMedCrossRefGoogle Scholar
  14. 14.
    Schellekens H. Immunogenicity of therapeutic proteins: clinical implications and future prospects. Clin Ther. 2002;24(11):1720–40.PubMedCrossRefGoogle Scholar
  15. 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. 16.
    Rosenberg A, editor. FDA Perspective on Immunogenicity Testing- A Risk Based Analysis. Bethesda, MD; 2003.Google Scholar
  17. 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.PubMedCrossRefGoogle Scholar
  18. 18.
    De Groot AS, Scott DW. Immunogenicity of protein therapeutics. Trends Immunol. 2007;28(11):482–90.PubMedCrossRefGoogle Scholar
  19. 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.PubMedGoogle Scholar
  20. 20.
    Schellekens H. Immunologic mechanisms of EPO-associated pure red cell aplasia. Best Pract Res Clin Haematol. 2005;18(3):473–80.PubMedCrossRefGoogle Scholar
  21. 21.
    Schernthaner G. Immunogenicity and allergenic potential of animal and human insulins. Diabetes Care. 1993;16 Suppl 3:155–65.PubMedCrossRefGoogle Scholar
  22. 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.PubMedCrossRefGoogle Scholar
  23. 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.PubMedCrossRefGoogle Scholar
  24. 24.
    Christian CL. Studies of aggregated γ-globulin: II effect in vivo. J Immunol. 1960;84(1):117–21.PubMedGoogle Scholar
  25. 25.
    Roskos LK, Davis CG, Schwab GM. The clinical pharmacology of therapeutic monoclonal antibodies. Drug Dev Res. 2004;61(3):108–20.CrossRefGoogle Scholar
  26. 26.
    Pendley C, Schantz A, Wagner C. Immunogenicity of therapeutic monoclonal antibodies. Curr Opin Mol Ther. 2003;5(2):172–9.PubMedGoogle Scholar
  27. 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.PubMedCrossRefGoogle Scholar
  28. 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.PubMedCrossRefGoogle Scholar
  29. 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.PubMedCrossRefGoogle Scholar
  30. 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. 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.PubMedCrossRefGoogle Scholar
  32. 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.PubMedCentralPubMedCrossRefGoogle Scholar
  33. 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.PubMedCrossRefGoogle Scholar
  34. 34.
    Brinks V, Jiskoot W, Schellekens H. Immunogenicity of therapeutic proteins: the use of animal models. Pharm Res. 2011;28(10):2379–85.PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Hwang WYK, Foote J. Immunogenicity of engineered antibodies. Methods (San Diego, CA, U S). 2005;36(1):3–10.CrossRefGoogle Scholar
  36. 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, 2010Google Scholar
  37. 37.
    Wang W. Protein aggregation and its inhibition in biopharmaceutics. Int J Pharm. 2005;289(1–2):1–30.PubMedCrossRefGoogle Scholar
  38. 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.PubMedCrossRefGoogle Scholar
  39. 39.
    PhEur 2.2.1. Clarity and degree of opalescence of liquids. European Directorate for the Quality of Medicine (EDQM). 7th edition; 2011.Google Scholar
  40. 40.
    PhEur 0169. Monograph “Water for injections”. European Directorate for the Quality of Medicine (EDQM). 7th edition; 2011.Google Scholar
  41. 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.PubMedCentralPubMedCrossRefGoogle Scholar
  42. 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.PubMedCrossRefGoogle Scholar
  43. 43.
    Petrovsky N, Aguilar JC. Vaccine adjuvants: current state and future trends. Immunol Cell Biol. 2004;82(5):488–96.PubMedCrossRefGoogle Scholar
  44. 44.
    Freitag AJ, Wittmann K, Winter G, Myschik J. The preparative use of flow field-flow fractionation. LCGC Europe. 2011;24(3):134.Google Scholar
  45. 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.PubMedCrossRefGoogle Scholar
  46. 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.PubMedCrossRefGoogle Scholar
  47. 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.PubMedCrossRefGoogle Scholar
  48. 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.CrossRefGoogle Scholar
  49. 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.PubMedCrossRefGoogle Scholar
  50. 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.PubMedGoogle Scholar
  51. 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.PubMedCrossRefGoogle Scholar
  52. 52.
    Vollmar D. Immunologie - Grundlagen und Wirkstoffe. 1st ed. München, Frankfurt am Main: Wissenschaftliche Verlagsgesellschaft mbH Stuttgart; 2005.Google Scholar
  53. 53.
    Haley PJ. Species differences in the structure and function of the immune system. Toxicology. 2003;188(1):49–71.PubMedCrossRefGoogle Scholar
  54. 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.PubMedCrossRefGoogle Scholar
  55. 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.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Angelika J. Freitag
    • 1
  • Maliheh Shomali
    • 2
  • Stylianos Michalakis
    • 5
  • Martin Biel
    • 5
  • Michael Siedler
    • 3
  • Zehra Kaymakcalan
    • 3
  • John F. Carpenter
    • 4
  • Theodore W. Randolph
    • 2
  • Gerhard Winter
    • 1
  • Julia Engert
    • 1
    Email author
  1. 1.Department of Pharmacy, Pharmaceutical Technology & BiopharmaceuticsLudwig-Maximilians-University MunichMunichGermany
  2. 2.Department of Chemical and Biological Engineering, Center for Pharmaceutical BiotechnologyUniversity of ColoradoBoulderUSA
  3. 3.AbbVie Bioresearch CenterWorcesterUSA
  4. 4.Department of Pharmaceutical Sciences, Center for Pharmaceutical BiotechnologyUniversity of Colorado Health Sciences CenterDenverUSA
  5. 5.Center for Integrated Protein Science Munich CiPSM and Department of Pharmacy – Center for Drug ResearchLudwig-Maximilians-Universität MünchenMunichGermany

Personalised recommendations