A Flow-Cytometry-Based Approach to Facilitate Quantification, Size Estimation and Characterization of Sub-visible Particles in Protein Solutions
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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.
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.
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.
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.
KEY WORDScross-β-sheet structures flow cytometry protein aggregates protein therapeutics sub-visible particles
- Aβ 1–40
Amyloid beta 1–40 peptide
4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt
Dulbecco’s Phosphate-Buffered Saline
- preparation Non-Prot
- preparation Prot
Protein particles without cross-ß-sheet structures
- preparation Prot-Crossß
Protein particles containing cross-ß-sheet structures
Recombinant human factor VIII
Transmission Electron Microscopy
Water For Injection
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.
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.
- 21.Ruiz L, Reyes N, Aroche K, Tolosa V, Simanca V, Rogríguez T, et al. Influence of packaging material on the liquid stability of interferon-alpha2b. J Pharm Sci. 2005;8(2):207–16.Google Scholar
- 38.EMA, Committee for Medicinal Products for Human Use. EMEA/CHMP/EWP/192217/2009 Guideline on Bioanalytical Method Validation. February 2012.Google Scholar
- 39.US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) and Center for Biologics Evaluation and Research (CBER). Draft guidance for industry: assay development for immunogenicity testing of therapeutic proteins. December 2009.Google Scholar
- 42.Wen ZQ, Torraca G, Yee CY, Li G. Investigation of contaminants in protein pharmaceuticals in pre-filled syringes by multiple micro-spectroscopies. Am Pharm Rev. 2007;10:101–7.Google Scholar
- 44.van der Pol E, Coumans FA, Grootemaat AE, Gardiner C, Sargent IL, Harrison P, et al. Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J Thromb Haemost. 2014;12(7):1182–92.PubMedCrossRefGoogle Scholar
- 45.Shapiro HM. Practical flow cytometry, 4th edition. Vienna, Austria:Wiley-Liss; 2003.Google Scholar
- 49.Fries A. Drug delivery of sensitive biopharmaceuticals with prefilled syringes. Drug Deliv Technol. 2009;9:22–7.Google Scholar