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

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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

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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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  5. Michael Siedler
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  6. Zehra Kaymakcalan
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  7. John F. Carpenter
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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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