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.
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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
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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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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