title: Fluorescence single particle tracking for the characterization of submicron protein aggregates in biological fluids and complex formulations.
abstract: OBJECTIVE
  To evaluate the potential of fluorescence single particle tracking (fSPT) for the characterization of submicron protein aggregates in human serum, plasma and formulations containing human serum albumin (HSA).
  
  
  METHODS
  A monoclonal IgG was covalently labeled with a fluorescent dye and cross-linked with glutaraldehyde. IgG aggregates and fluorescent beads of 0.1 μm (control) were diluted in buffer, serum and plasma, and their size distributions were analyzed by fSPT and nanoparticle tracking analysis (NTA). In a separate experiment, IgG and HSA, fluorescently labeled with different dyes, were mixed and subjected to heat stress. The stressed sample was analyzed by fSPT using a dual color mode and by NTA.
  
  
  RESULTS
  The accuracy and precision of fSPT proved to be comparable to NTA. fSPT was able to successfully measure all the samples in buffer, serum and plasma. The average size of the cross-linked protein aggregates showed a slight increase in biological fluids. Moreover, fSPT analysis showed that a significant proportion of the aggregates formed by subjecting an IgG/HSA mixture to heat stress were composed of both proteins.
  
  
  CONCLUSIONS
  fSPT is a powerful technique for the characterization of submicron protein aggregates in biological fluids and complex formulations.
authors.affiliation: Division of Drug Delivery Technology Leiden/Amsterdam Center for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands.
authors.forename: Vasco
authors.initials: V
authors.lastname: Filipe
authors.forename: Robert
authors.initials: R
authors.lastname: Poole
authors.forename: Marika
authors.initials: M
authors.lastname: Kutscher
authors.forename: Katrien
authors.initials: K
authors.lastname: Forier
authors.forename: Kevin
authors.initials: K
authors.lastname: Braeckmans
authors.forename: Wim
authors.initials: W
authors.lastname: Jiskoot
mesh_terms: D003432:Cross-Linking Reagents; D005079:Excipients / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis*; D053758:Nanoparticles / Q000032:analysis*; D010316:Particle Size; D010949:Plasma / Q000737:chemistry*; D011487:Protein Conformation; D044967:Serum / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D005079:Excipients; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D012709:Serum Albumin