title: A Flow-Cytometry-Based Approach to Facilitate Quantification, Size Estimation and Characterization of Sub-visible Particles in Protein Solutions.
abstract: OBJECTIVE
  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.
authors: Baxter Innovation GmbH, Industriestrasse 72, A-1220, Vienna, Austria.
authors: Christian
authors: C
authors: Lubich
authors: Mantas
authors: M
authors: Malisauskas
authors: Thomas
authors: T
authors: Prenninger
authors: Thomas
authors: T
authors: Wurz
authors: Peter
authors: P
authors: Matthiessen
authors: Peter L
authors: PL
authors: Turecek
authors: Friedrich
authors: F
authors: Scheiflinger
authors: Birgit M
authors: BM
authors: Reipert
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D002138:Calibration; D005434:Flow Cytometry / Q000379:methods; D010316:Particle Size; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D012996:Solutions / Q000737:chemistry*
chemical_list: D000817:Anilino Naphthalenesulfonates; D011506:Proteins; D012996:Solutions; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate)