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

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 polyso...

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Detalles Bibliográficos
Autores principales: Hawe, Andrea, Filipe, Vasco, Jiskoot, Wim
Formato: Texto
Lenguaje:English
Publicado: Springer US 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2812426/
https://www.ncbi.nlm.nih.gov/pubmed/20041280
http://dx.doi.org/10.1007/s11095-009-0020-2
Descripción
Sumario: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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11095-009-0020-2) contains supplementary material, which is available to authorized users.