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In Vivo Fluorescence Imaging of IgG1 Aggregates After Subcutaneous and Intravenous Injection in Mice

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ABSTRACT

Purpose

To monitor the biodistribution of IgG1 aggregates upon subcutaneous (SC) and intravenous (IV) administration in mice and measure their propensity to stimulate an early immune response.

Methods

A human mAb (IgG1) was fluorescently labeled, aggregated by agitation stress and injected in SKH1 mice through SC and IV routes. The biodistribution of monomeric and aggregated formulations was monitored over 47 days by fluorescence imaging and the early immune response was measured by quantifying the level of relevant cytokines in serum using a Bio-plex assay.

Results

The aggregates remained at the SC injection site for a longer time than monomers but after entry into the systemic circulation disappeared faster than monomers. Upon IV administration, both monomers and aggregates spread rapidly throughout the circulation, and a strong accumulation in the liver was observed for both species. Subsequent removal from the circulation was faster for aggregates than monomers. No accumulation in lymph nodes was observed after SC or IV administration. Administration of monomers and aggregates induced similar cytokine levels, but SC injection resulted in higher cytokine levels than IV administration.

Conclusion

These results show differences in biodistribution and residence time between IgG1 aggregates and monomers. The long residence time of aggregates at the SC injection site, in conjunction with elevated cytokine levels, may contribute to an enhanced immunogenicity risk of SC injected aggregates compared to that of monomers.

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors acknowledge Els van Beelen for her help with the Bio-Plex assay.

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Authors and Affiliations

  1. Division of Drug Delivery Technology Leiden Academic Center for Drug Research, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands

    Vasco Filipe & Wim Jiskoot

  2. Department of Radiology, Leiden University Medical Centre, Leiden, The Netherlands

    Ivo Que & Clemens Löwik

  3. Department of Pharmaceutical Sciences, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, 80045, USA

    John F. Carpenter

Authors
  1. Vasco Filipe
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Corresponding author

Correspondence to Wim Jiskoot.

Electronic Supplementary Material

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Appendix A

Representative fluorescence images over time of monomeric and aggregated 800CW-IgG1 upon SC injection in SKH1 mice. Five mice per group were imaged and used for fluorescence quantification purposes. Dorsal and ventral images of the same mouse were obtained throughout a time period of 47 days after injection. The fluorescence threshold of the first time points (upper panel) was optimized to not over-expose the first time point (Settings 1). The fluorescence threshold of the last time points (lower panel) was optimized to show the maximum fluorescence possible before auto-fluorescence was reached (Settings 2). (JPEG 46 kb)

High resolution image (TIFF 6112 kb)

Appendix B

Representative fluorescence images over time of monomeric and aggregated 800CW-IgG1 upon IV injection in SKH1 mice. Five mice per group were imaged and used for fluorescence quantification purposes. Dorsal and ventral images of the same mouse were obtained throughout a time period of 47 days after injection. The fluorescence threshold of the first time points (upper panel) was optimized to not over-expose the first time point (Settings 1). The fluorescence threshold of the last time points (lower panel) was optimized to show the maximum fluorescence possible before auto-fluorescence was reached (Settings 2). (JPEG 56 kb)

High resolution image (TIFF 6115 kb)

Appendix C

Fluorescence images over time of non-reactive free 800CW (green) and RD680 (red) dyes upon SC and IV injection in SKH1 mice. The upper panel corresponds to SC injections and the lower panel to an IV injection. One mouse per group was imaged. Dorsal and ventral images were obtained throughout a time period of 3 days after injection. (JPEG 51 kb)

High resolution image (TIFF 13507 kb)

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Filipe, V., Que, I., Carpenter, J.F. et al. In Vivo Fluorescence Imaging of IgG1 Aggregates After Subcutaneous and Intravenous Injection in Mice. Pharm Res 31, 216–227 (2014). https://doi.org/10.1007/s11095-013-1154-9

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  • DOI: https://doi.org/10.1007/s11095-013-1154-9

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