Fluorescent molecular rotors as dyes to characterize polysorbate-containing IgG formulations

Pharm Res. 2010 Feb;27(2):314-26. doi: 10.1007/s11095-009-0020-2. Epub 2009 Dec 30.

Abstract

Purpose: The aim was to evaluate fluorescent molecular rotors (DCVJ and CCVJ), which are mainly sensitive to viscosity, for the characterization of polysorbate-containing IgG formulations and compare them to the polarity-sensitive dyes ANS, Bis-ANS and Nile Red.

Methods: IgG formulations with polysorbate 20 or 80 were stressed below the aggregation temperature and analyzed by steady-state and time-resolved fluorescence and by HP-SEC with UV and fluorescent dye detection (Bis-ANS and CCVJ). Furthermore, commercial protein preparations of therapeutic proteins (Enbrel 50 mg, Humira 40 mg and MabThera 100 mg) were aggregated accordingly and analyzed with CCVJ fluorescence and HP-SEC.

Results: Contrarily to (Bis-)ANS and Nile Red, the molecular rotors DCVJ and CCVJ showed low background fluorescence in polysorbate-containing buffers. Time-resolved fluorescence experiments confirmed the steady-state fluorescence data. Both DCVJ and CCVJ showed enhanced fluorescence intensity for aggregated IgG formulations and were suitable for the characterization of polysorbate-containing IgG formulations in steady-state fluorescence and HP-SEC with dye detection (CCVJ). CCVJ was capable of detecting thermally induced aggregation in the commercial polysorbate-containing products Enbrel 50 mg, Humira 40 mg and MabThera 100 mg.

Conclusion: Fluorescent molecular rotors are suitable probes to detect aggregation in polysorbate-containing IgG formulations.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / chemistry
  • Chemistry, Pharmaceutical
  • Fluorescent Dyes / chemistry*
  • Humans
  • Immunoglobulin G / chemistry*
  • Molecular Probes / chemistry*
  • Polysorbates / chemistry*
  • Recombinant Proteins / chemistry
  • Spectrometry, Fluorescence

Substances

  • Antibodies, Monoclonal
  • Fluorescent Dyes
  • Immunoglobulin G
  • Molecular Probes
  • Polysorbates
  • Recombinant Proteins