Related Experiment Video
Updated: Apr 16, 2026
Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry
Published on: April 6, 2022
A Flow-Cytometry-Based Approach to Facilitate Quantification, Size Estimation and Characterization of Sub-visible
Christian Lubich1, Mantas Malisauskas, Thomas Prenninger
1Baxter Innovation GmbH, Industriestrasse 72, A-1220, Vienna, Austria.
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.
