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A Flow-Cytometry-Based Approach to Facilitate Quantification, Size Estimation and Characterization of Sub-visible Particles in Protein Solutions

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Abstract

Purpose

Sub-visible particles were shown to facilitate unwanted immunogenicity of protein therapeutics. To understand the root cause of this phenomenon, a comprehensive analysis of these particles is required. We aimed at establishing a flow-cytometry-based technology to analyze the amount, size distribution and nature of sub-visible particles in protein solutions.

Methods

We adjusted the settings of a BD FACS Canto II by tuning the forward scatter and the side scatter detectors and by using size calibration beads to facilitate the analysis of particles with sizes below 1 μM. We applied a combination of Bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt) and DCVJ (9-(2,2-dicyanovinyl)julolidine) to identify specific characteristics of sub-visible particles.

Results

The FACS technology allows the analysis of particles between 0.75 and 10 μm in size, requiring relatively small sample volumes. Protein containing particles can be distinguished from non-protein particles and cross-β-sheet structures contained in protein particles can be identified.

Conclusions

The FACS technology provides robust and reproducible results with respect to number, size distribution and specific characteristics of sub-visible particles between 0.75 and 10 μm in size. Our data for number and size distribution of particles is in good agreement with results obtained with the state-of-the-art technology micro-flow imaging.

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Abbreviations

Aβ 1–40:

Amyloid beta 1–40 peptide

Bis-ANS:

4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt

DCVJ:

9-(2,2-dicyanovinyl)julolidine

DMSO:

Dimethyl sulfoxide

D-PBS:

Dulbecco’s Phosphate-Buffered Saline

preparation Non-Prot:

Non-protein particles

preparation Prot:

Protein particles without cross-ß-sheet structures

preparation Prot-Crossß:

Protein particles containing cross-ß-sheet structures

rFVIII:

Recombinant human factor VIII

TEM:

Transmission Electron Microscopy

ThT:

Thioflavin T

WFI:

Water For Injection

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors thank Elise Langdon-Neuner and Karima Benamara for editing the manuscript.

This work was supported by Baxter Innovation GmbH. C.L., M.M., T.P., T.W., P.M., P.L.T., F.S. and B.M.R. are employees of Baxter Innovation GmbH.

Authors Contribution

C. L. designed research, performed flow cytometric analysis, analyzed and interpreted data, and wrote the paper; M.M. designed research, analyzed and interpreted data, and wrote the paper; T.P. performed flow cytometric analysis, and analyzed and interpreted data, T.W. performed flow cytometric analysis of the method validation; P.M. performed, analyzed and interpreted micro flow imaging data; P.L.T. interpreted data; F.S. interpreted data; B.M.R. designed research, analyzed and interpreted data, and wrote the paper.

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Authors and Affiliations

  1. Baxter Innovation GmbH, Industriestrasse 72, A-1220, Vienna, Austria

    Christian Lubich, Mantas Malisauskas, Thomas Prenninger, Thomas Wurz, Peter Matthiessen, Peter L. Turecek, Friedrich Scheiflinger & Birgit M. Reipert

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  1. Christian Lubich
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Correspondence to Birgit M. Reipert.

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Lubich, C., Malisauskas, M., Prenninger, T. et al. A Flow-Cytometry-Based Approach to Facilitate Quantification, Size Estimation and Characterization of Sub-visible Particles in Protein Solutions. Pharm Res 32, 2863–2876 (2015). https://doi.org/10.1007/s11095-015-1669-3

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