Skip to main content
Log in

Discrimination Between Silicone Oil Droplets and Protein Aggregates in Biopharmaceuticals: A Novel Multiparametric Image Filter for Sub-visible Particles in Microflow Imaging Analysis

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

ABSTRACT

Purpose

Accurate monitoring of the sub-visible particle load in protein biopharmaceuticals is increasingly important to drug development. Manufacturers are expected to characterize and control sub-visible protein particles in their products due to their potential immunogenicity. Light obscuration, the most commonly used analytical tool to count microscopic particles, does not allow discrimination between potentially harmful protein aggregates and harmless pharmaceutical components, e.g. silicone oil, commonly present in drug products. Microscopic image analysis in flow-microscopy techniques allows not only counting, but also classification of sub-visible particles based on morphology. We present a novel approach to define software filters for analysis of particle morphology in flow-microscopic images enhancing the capabilities of flow-microscopy.

Methods

Image morphology analysis was applied to analyze flow-microscopy data from experimental test sets of protein aggregates and silicone oil suspensions.

Results

A combination of four image morphology parameters was found to provide a reliable basis for automatic distinction between silicone oil droplets and protein aggregates in protein biopharmaceuticals resulting in low misclassification errors.

Conclusions

A novel, custom-made software filter for discrimination between proteinaceous particles and silicone oil droplets in flow-microscopy imaging analysis was successfully developed.

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

Access this article

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

Similar content being viewed by others

Abbreviations

AR:

aspect ratio

MFI:

microflow imaging

NA:

numerical aperture

PBS:

Phosphate buffer saline

TFA:

trifluoroacetic acid

REFERENCES

  1. Rosenberg AS. Effects of protein aggregates: an immunologic perspective. AAPS J. 2006;8(3):E501–7.

    Article  PubMed  Google Scholar 

  2. Carpenter JF, Randolph TW, Jiskoot W, Crommelin DJ, Middaugh CR, Winter G, et al. Overlooking subvisible particles in therapeutic protein products: gaps that may compromise product quality. J Pharm Sci. 2009;98(4):1201–5.

    Article  PubMed  CAS  Google Scholar 

  3. Singh SK, Afonina N, Awwad M, Bechtold-Peters K, Blue JT, Chou D, et al. An industry perspective on the monitoring of subvisible particles as a quality attribute for protein therapeutics. J Pharm Sci. 2010;99(8):3302–21.

    Article  PubMed  CAS  Google Scholar 

  4. 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(8):3343–61.

    Article  PubMed  CAS  Google Scholar 

  5. Liu L, Ammar DA, Ross LA, Mandava N, Kahook MY, Carpenter JF. Silicone oil microdroplets and protein aggregates in repackaged bevacizumab and ranibizumab: effects of long-term storage and product mishandling. Invest Ophthalmol Vis Sci. 2011;52(2):1023–34.

    Article  PubMed  CAS  Google Scholar 

  6. 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(3):455–64.

    Article  PubMed  CAS  Google Scholar 

  7. Sharma D, Oma P, Krishnan S. Silicone micro-droplets in protein formulations—detection and enumeration. Pharm Technol. 2009;33(4):74–9.

    CAS  Google Scholar 

  8. Mahler HC, Friess W, Grauschopf U, Kiese S. Protein aggregation: pathways, induction factors and analysis. J Pharm Sci. 2009;98(9):2909–34.

    Article  PubMed  CAS  Google Scholar 

  9. Huang CT, Sharma D, Oma P, Krishnamurthy R. Quantitation of protein particles in parenteral solutions using micro-flow imaging. J Pharm Sci. 2009;98(9):3058–71.

    Article  PubMed  CAS  Google Scholar 

Download references

ACKNOWLEDGMENTS & DISCLOSURES

We would like to thank Christian Reithmaier for his support with MS Excel.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Atanas V. Koulov.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOCX 145 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Strehl, R., Rombach-Riegraf, V., Diez, 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 29, 594–602 (2012). https://doi.org/10.1007/s11095-011-0590-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-011-0590-7

KEY WORDS

Navigation