Investigation of the Immunogenicity of Different Types of Aggregates of a Murine Monoclonal Antibody in Mice
- 1.1k Downloads
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.
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.
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.
This study shows that not all protein drug aggregates are equally immunogenic.
Key WordsImmunogenicity Monoclonal antibody Protein aggregates Protein particles Wild-type mice
Asymmetric flow field flow fractionation
Attenuated total reflection
Area under the curve
Enzyme-linked immunosorbent assay
Formazine nephelometric units
Multi angle laser light scattering
Phosphate buffered saline
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.
- 7.Swanson SJ, editor. Immunogenicity of Therapeutic Proteins. Hoboken: Wiley; 2010.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
- 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.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.PubMedCrossRefGoogle 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
- 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
- 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.PhEur 0169. Monograph “Water for injections”. European Directorate for the Quality of Medicine (EDQM). 7th edition; 2011.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.Google Scholar
- 52.Vollmar D. Immunologie - Grundlagen und Wirkstoffe. 1st ed. München, Frankfurt am Main: Wissenschaftliche Verlagsgesellschaft mbH Stuttgart; 2005.Google Scholar