Fluorescent Molecular Rotors as Dyes to Characterize Polysorbate-Containing IgG Formulations

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. 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 (EnbrelA (R) 50 mg, HumiraA (R) 40 mg and MabTheraA (R) 100 mg) were aggregated accordingly and analyzed with CCVJ fluorescence and HP-SEC. 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 EnbrelA (R) 50 mg, HumiraA (R) 40 mg and MabTheraA (R) 100 mg. Fluorescent molecular rotors are suitable probes to detect aggregation in polysorbate-containing IgG formulations.