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Physical Characterization and Innate Immunogenicity of Aggregated Intravenous Immunoglobulin (IGIV) in an In Vitro Cell-Based Model

Pharmaceutical Research volume 33, pages 1736–1751 (2016)Cite this article

Abstract

Purpose

To investigate in vitro the innate immune response to accelerated stress-induced aggregates of intravenous immunoglobulin (IGIV) using a well-defined human cell-line model, and to correlate the innate response to physical properties of the aggregates.

Methods

IGIV aggregates were prepared by applying various accelerated stress methods, and particle size, count and structure were characterized. Immune cell activation as tracked by inflammatory cytokines released in response to aggregates was evaluated in vitro using peripheral blood mononuclear cells (PBMC), primary monocytes and immortalized human monocyte-like cell lines.

Results

IGIV aggregates produced by mechanical stress induced higher cytokine release by PBMC and primary monocytes than aggregates formed by other stresses. Results with the monocytic cell line THP-1 paralleled trends in PBMC and primary monocytes. Effects were dose-dependent, enhanced by complement opsonization, and partially inhibited by blocking toll-like receptors (TLR2 and TLR4) and to a lesser extent by blocking Fc gamma receptors (FcγRs).

Conclusions

Stress-induced IGIV aggregates stimulate a dose-dependent cytokine response in human monocytes and THP-1 cells, mediated in part by TLRs, FcγRs and complement opsonization. THP-1 cells resemble primary monocytes in many respects with regard to tracking the innate response to IgG aggregates. Accordingly, the measurement of inflammatory cytokines released by THP-1 cells provides a readily accessible assay system to screen for the potential innate immunogenicity of IgG aggregates. The results also highlight the role of aggregate structure in interacting with the different receptors mediating innate immunity.

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Abbreviations

APC:

Antigen presenting cell

C3:

Complement 3

CBA:

Cytometric bead array

CD:

Circular dichroism

FcγR:

Fc gamma receptor

HPLC:

High performance liquid chromatography

IC:

Immune complex

IGIV:

Intravenous immunoglobulin

NTA:

Nanoparticle tracking analysis

PAMP:

Pathogen associated molecular patterns

PBMC:

Peripheral blood mononuclear cell

RMM:

Resonant mass measurement

SEC:

Size exclusion chromatography

TLR:

Toll-like receptor

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors gratefully thank Daniela Verthelyi and Jennifer Reed for critically reading the manuscript and providing valuable suggestions and comments. The authors are also thankful to Nancy Eller for useful discussions and to Eunbi Cho for assisting with cell-based assays. This work was funded by FDA contract HHSF223201310233C (PI: E. M. Topp). This project was supported in part by an appointment (JK) to the Research Participation Program at the Center for Biologics Evaluation and Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. The authors have no conflicts of interest to declare.

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Author notes

  1. J. Kotarek

    Present address: Brand Institute, Rockville, Maryland, USA

Authors and Affiliations

  1. Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, 47906, West Lafayette, Indiana, USA

    E. M. Moussa & E. M. Topp

  2. Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland, USA

    J. Kotarek & E. Marszal

  3. Department of Microbiology and Immunology, School of Medicine, Indiana University, Indianapolis, Indiana, USA

    J. S. Blum

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Correspondence to E. M. Topp.

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Supplementary Fig. 1

SDS-PAGE of different aggregated IGIV samples. (a) Non-reduced conditions. (b) Reduced conditions. M, protein marker; UN, unstressed IGIV; HT, heated IGIV; OX, oxidized IGIV; SH, shaken IGIV; ST, stirred IGIV. (JPG 124 kb)

Supplementary Fig. 2

Dose-response of primary monocytes to stirring-induced IGIV aggregates. (a-e) individual cytokines response to aggregates in primary monocytes. Bars represent 1 standard deviation of the mean response of the donors (n = 4). Each colored closed circle represents the average response of an individual donor (n = 5). SI, stimulation index over the response to unstressed IGIV. The x-axis represents the number of micro particles as determined by flow imaging per one million cells. (JPG 705 kb)

Supplementary Fig. 3

Dose-response of THP-1 cells to stirring-induced IGIV aggregates. (a-e) individual cytokine response to aggregates in THP-1 cells. Bars represent 1 standard deviation of the mean response of cell batches (n = 3). Each colored closed circle represents the average response of a different THP-1 cell batch. SI, stimulation index over the response to unstressed IGIV. The x-axis represents the number of particles as determined by flow imaging per one million cells. (JPG 665 kb)

Supplementary Fig. 4

Expression of innate immunity receptors on the surface of THP-1 cells. x-axes represent the fluorescence intensity measured by flow cytometry using the appropriate channel for the fluorescent dye on each antibody, and y-axes represent the counts of labeled cells. Red curves represent unlabeled controls, whereas blue curves represent labeled cells. (JPG 98 kb)

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Moussa, E.M., Kotarek, J., Blum, J.S. et al. Physical Characterization and Innate Immunogenicity of Aggregated Intravenous Immunoglobulin (IGIV) in an In Vitro Cell-Based Model. Pharm Res 33, 1736–1751 (2016). https://doi.org/10.1007/s11095-016-1914-4

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