Skip to main content
SpringerLink
Log in

Flow Imaging Microscopy for Protein Particle Analysis—A Comparative Evaluation of Four Different Analytical Instruments

  • Research Article
  • Published:
The AAPS Journal Aims and scope Submit manuscript

Abstract

Flow imaging microscopy was introduced as a technique for protein particle analysis a few years ago and has strongly gained in importance ever since. The aim of the present study was a comparative evaluation of four of the most relevant flow imaging microscopy systems for biopharmaceuticals on the market: Micro-Flow Imaging (MFI)4100, MFI5200, Flow Cytometer And Microscope (FlowCAM) VS1, and FlowCAM PV. Polystyrene standards, particles generated from therapeutic monoclonal antibodies, and silicone oil droplets were analyzed by all systems. The performance was critically assessed regarding quantification, characterization, image quality, differentiation of protein particles and silicone oil droplets, and handling of the systems. The FlowCAM systems, especially the FlowCAM VS1, showed high-resolution images. The FlowCAM PV system provided the most precise quantification of particles of therapeutic monoclonal antibodies, also under impaired optical conditions by an increased refractive index of the formulation. Furthermore, the most accurate differentiation of protein particles and silicone oil droplets could be achieved with this instrument. The MFI systems provided excellent size and count accuracy (evaluated with polystyrene standards) especially the MFI5200 system. This instrument also showed very good performance for protein particles, also in case of an increased refractive index of the formulation. Both MFI systems were easier to use and appeared more standardized regarding measurement and data analysis as compared to the FlowCAM systems. Our study shows that the selection of the appropriate flow imaging microscopy system depends strongly on the main output parameters of interest and it is recommended to decide based on the intended application.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

REFERENCES

  1. Carpenter JF, Randolph TW, Jiskoot W, Crommelin DJA, Middaugh CR, Winter G, et al. Overlooking subvisible particles in therapeutic protein products: gaps that may compromise product quality. J Pharm Sci. 2009;98:1201–5.

    Article  PubMed  CAS  Google Scholar 

  2. Carpenter J, Cherney B, Lubinecki A, Ma S, Marszal E, Mire-Sluis A, et al. Meeting report on protein particles and immunogenicity of therapeutic proteins: filling in the gaps in risk evaluation and mitigation. Biologicals. 2010;38:602–11.

    Article  PubMed  Google Scholar 

  3. Hawe A, Wiggenhorn M, van de Weert M, Garbe JHO, Mahler H-C, Jiskoot W. Forced degradation of therapeutic proteins. J Pharm Sci. 2012;101:895–913.

    Article  PubMed  CAS  Google Scholar 

  4. Narhi LO, Schmit J, Bechtold-Peters K, Sharma D. Classification of protein aggregates. J Pharm Sci. 2012;101:493–8.

    Article  PubMed  CAS  Google Scholar 

  5. Rosenberg AS. Effects of protein aggregates: an immunologic perspective. AAPS J. 2006;8:E501–7.

    Article  PubMed  Google Scholar 

  6. Ph.Eur. 2.9.19, Pharmacopoea Europaea, 7th ed. 2010. Particulate contamination: sub-visible particles. European Directorate for the Quality of Medicine (EDQM).

  7. USP<788>, United States Pharmacopeia, USP35-NF30, 2012. Particulate matter in injections. United States Pharmacopeial convention.

  8. Kirshner S. Regulatory expectations for analysis of aggregates and particles. Talk at Workshop on Protein Aggregation and Immunogenicity, Breckenridge, Colorado. 12 July 2012.

  9. Food US, Administration D. Guidance for industry. Immunogenicity assessment for therapeutic protein products (draft guidance). Silver Spring, MD, USA: FDA; 2013.

    Google Scholar 

  10. Zölls S, Tantipolphan R, Wiggenhorn M, Winter G, Jiskoot W, Friess W, et al. Particles in therapeutic protein formulations, part 1: overview of analytical methods. J Pharm Sci. 2012;101:914–35.

    Article  PubMed  Google Scholar 

  11. Burg TP, Godin M, Knudsen SM, Shen W, Carlson G, Foster JS, et al. Weighing of biomolecules, single cells and single nanoparticles in fluid. Nature. 2007;446:1066–9.

    Article  PubMed  CAS  Google Scholar 

  12. Narhi LO. AAPS update on USP expert committee for sub-visible particle analysis. Newsletter of the AAPS Aggregation and Biological Relevance Focus Group. 2012;3(2).

  13. Demeule B, Messick S, Shire SJ, Liu J. Characterization of particles in protein solutions: reaching the limits of current technologies. AAPS J. 2010;12:708–15.

    Article  PubMed  CAS  Google Scholar 

  14. Sharma DK, Oma P, Pollo MJ, Sukumar M. Quantification and characterization of subvisible proteinaceous particles in opalescent mAb formulations using micro-flow imaging. J Pharm Sci. 2010;99:2628–42.

    PubMed  CAS  Google Scholar 

  15. Wuchner K, Büchler J, Spycher R, Dalmonte P, Volkin DB. Development of a microflow digital imaging assay to characterize protein particulates during storage of a high concentration IgG1 monoclonal antibody formulation. J Pharm Sci. 2010;99:3343–61.

    Article  PubMed  CAS  Google Scholar 

  16. Joubert MK, Luo Q, Nashed-Samuel Y, Wypych J, Narhi LO. Classification and characterization of therapeutic antibody aggregates. JBC. 2011;286:25118–33.

    Article  CAS  Google Scholar 

  17. Barnard JG, Babcock K, Carpenter JF. Characterization and quantitation of aggregates and particles in interferon-β products: potential links between product quality attributes and immunogenicity. J Pharm Sci. 2012;102:915–28.

    Article  PubMed  Google Scholar 

  18. Barnard JG, Singh S, Randolph TW, Carpenter JF. Subvisible particle counting provides a sensitive method of detecting and quantifying aggregation of monoclonal antibody caused by freeze-thawing: insights into the roles of particles in the protein aggregation pathway. J Pharm Sci. 2011;100:492–503.

    Article  PubMed  CAS  Google Scholar 

  19. Patel AR, Lau D, Liu J. Quantification and characterization of micrometer and submicrometer subvisible particles in protein therapeutics by use of a suspended microchannel resonator. Anal Chem. 2012;84(15):6833–40.

    Article  PubMed  CAS  Google Scholar 

  20. Sharma DK, King D, Oma P, Merchant C. Micro-flow imaging: flow microscopy applied to sub-visible particulate analysis in protein formulations. AAPS J. 2010;12:455–64.

    Article  PubMed  CAS  Google Scholar 

  21. Brown L. Characterizing biologics using dynamic imaging particle analysis. BioPharm Int. 2011;24(8):s4–9.

    Google Scholar 

  22. Weinbuch D, Zölls S, Wiggenhorn M, Friess W, Winter G, Jiskoot W, et al. Micro-flow imaging and resonant mass measurement (Archimedes)—complimentary methods to quantitatively differentiate protein particles and silicone oil droplets. J Pharm Sci. 2013;102:2152–65.

    Article  PubMed  CAS  Google Scholar 

  23. Strehl R, Rombach-Riegraf V, Diez M, Egodage K, Bluemel M, Jeschke M, et al. Discrimination between silicone oil droplets and protein aggregates in biopharmaceuticals: a novel multiparametric image filter for sub-visible particles in microflow imaging analysis. Pharm Res. 2012;29(2):594–602.

    Article  PubMed  CAS  Google Scholar 

  24. Sharma BDK, Oma P, Krishnan S. Silicone microdroplets in protein formulations—detection and enumeration. Pharm Technol. 2009;33:74–9.

    CAS  Google Scholar 

  25. Huang C-T, Sharma D, Oma P, Krishnamurthy R. Quantitation of protein particles in parenteral solutions using micro-flow imaging. J Pharm Sci. 2009;98:3058–71.

    Article  PubMed  CAS  Google Scholar 

  26. Zölls S, Gregoritza M, Tantipolphan R, Wiggenhorn M, Winter G, Friess W, et al. How subvisible particles become invisible—relevance of the refractive index for protein particle analysis. J Pharm Sci. 2013;102:1434–46.

    Article  PubMed  Google Scholar 

  27. Wilson GA, Manning MC. Flow imaging: moving toward best practices for subvisible particle quantitation in protein products. J Pharm Sci. 2013;102:1133–4.

    Article  PubMed  CAS  Google Scholar 

Download references

ACKNOWLEDGMENTS

We thank Axel Wilde from Anasysta (distributor of Fluid Imaging in Germany) and Josh Geib from Fluid Imaging for providing access to the FlowCAM systems and Dave Palmlund from Fluid Imaging for helpful comments to the manuscript.

Author information

Authors and Affiliations

  1. Coriolis Pharma, Am Klopferspitz 19, 82152, Martinsried, Germany

    Sarah Zölls, Daniel Weinbuch, Michael Wiggenhorn & Andrea Hawe

  2. Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig Maximilian University, Butenandtstr. 5-13, 81377, Munich, Germany

    Sarah Zölls, Gerhard Winter & Wolfgang Friess

  3. Leiden Academic Centre for Drug Research, Division of Drug Delivery Technology, Leiden University, 2300, RA, Leiden, The Netherlands

    Daniel Weinbuch & Wim Jiskoot

Authors
  1. Sarah Zölls
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel Weinbuch
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael Wiggenhorn
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gerhard Winter
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wolfgang Friess
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wim Jiskoot
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Andrea Hawe
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Andrea Hawe.

Additional information

Sarah Zölls and Daniel Weinbuch equally contributed to this paper.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

ESM 1

DOCX 68 kb

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zölls, S., Weinbuch, D., Wiggenhorn, M. et al. Flow Imaging Microscopy for Protein Particle Analysis—A Comparative Evaluation of Four Different Analytical Instruments. AAPS J 15, 1200–1211 (2013). https://doi.org/10.1208/s12248-013-9522-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1208/s12248-013-9522-2

KEY WORDS

Search

Navigation