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

Hawe A, Filipe V, Jiskoot W - Pharm. Res. (2009)

Bottom Line: Time-resolved fluorescence experiments confirmed the steady-state fluorescence data.CCVJ was capable of detecting thermally induced aggregation in the commercial polysorbate-containing products Enbrel 50 mg, Humira 40 mg and MabThera 100 mg.Fluorescent molecular rotors are suitable probes to detect aggregation in polysorbate-containing IgG formulations.

View Article: PubMed Central - PubMed

Affiliation: Division of Drug Delivery Technology, Leiden/Amsterdam Center for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands. ahawe@chem.leidenuniv.nl

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.

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Related in: MedlinePlus

Representative steady state fluorescence spectra of 5 µM DCVJ (A), 5 μM CCVJ (B) and 5 μM Bis-ANS (C) in 100 mM phosphate pH 7.2 (buffer), 10% (w/v) sucrose, methanol, ethanol and glycerol.
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Fig1: Representative steady state fluorescence spectra of 5 µM DCVJ (A), 5 μM CCVJ (B) and 5 μM Bis-ANS (C) in 100 mM phosphate pH 7.2 (buffer), 10% (w/v) sucrose, methanol, ethanol and glycerol.

Mentions: In order to identify suitable dyes for the analysis of polysorbate-containing formulations, a number of dyes were compared for their sensitivity to polarity and viscosity. To do so, the fluorescence emission of the dyes was measured in 100 mM phosphate pH 7.2 (dielectric constant ε = 80.1, viscosity at 20°C η  = 1 mPa s), 10% (w/v) sucrose (ε = 80.1, η  = 1.2 mPa s), ethanol (ε = 25, η  = 1.2 mPa s), methanol (ε = 33.6, η  = 0.59 mPa s) and glycerol (ε = 42.5, η  = 945 mPa s) (Fig. 1). A considerable intensity increase combined with a blue shift of at most 32 nm was measured for Bis-ANS in ethanol and methanol. In glycerol, Bis-ANS fluorescence intensity did not increase as much, and the blue shift was only 10 nm. DCVJ and CCVJ exhibited significantly higher fluorescence intensities in glycerol, but unchanged (CCVJ) or slightly increased (DCVJ) intensities in ethanol and methanol. The blue shift in organic solvents was higher than the one in glycerol.Fig. 1


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

Hawe A, Filipe V, Jiskoot W - Pharm. Res. (2009)

Representative steady state fluorescence spectra of 5 µM DCVJ (A), 5 μM CCVJ (B) and 5 μM Bis-ANS (C) in 100 mM phosphate pH 7.2 (buffer), 10% (w/v) sucrose, methanol, ethanol and glycerol.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2812426&req=5

Fig1: Representative steady state fluorescence spectra of 5 µM DCVJ (A), 5 μM CCVJ (B) and 5 μM Bis-ANS (C) in 100 mM phosphate pH 7.2 (buffer), 10% (w/v) sucrose, methanol, ethanol and glycerol.
Mentions: In order to identify suitable dyes for the analysis of polysorbate-containing formulations, a number of dyes were compared for their sensitivity to polarity and viscosity. To do so, the fluorescence emission of the dyes was measured in 100 mM phosphate pH 7.2 (dielectric constant ε = 80.1, viscosity at 20°C η  = 1 mPa s), 10% (w/v) sucrose (ε = 80.1, η  = 1.2 mPa s), ethanol (ε = 25, η  = 1.2 mPa s), methanol (ε = 33.6, η  = 0.59 mPa s) and glycerol (ε = 42.5, η  = 945 mPa s) (Fig. 1). A considerable intensity increase combined with a blue shift of at most 32 nm was measured for Bis-ANS in ethanol and methanol. In glycerol, Bis-ANS fluorescence intensity did not increase as much, and the blue shift was only 10 nm. DCVJ and CCVJ exhibited significantly higher fluorescence intensities in glycerol, but unchanged (CCVJ) or slightly increased (DCVJ) intensities in ethanol and methanol. The blue shift in organic solvents was higher than the one in glycerol.Fig. 1

Bottom Line: Time-resolved fluorescence experiments confirmed the steady-state fluorescence data.CCVJ was capable of detecting thermally induced aggregation in the commercial polysorbate-containing products Enbrel 50 mg, Humira 40 mg and MabThera 100 mg.Fluorescent molecular rotors are suitable probes to detect aggregation in polysorbate-containing IgG formulations.

View Article: PubMed Central - PubMed

Affiliation: Division of Drug Delivery Technology, Leiden/Amsterdam Center for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands. ahawe@chem.leidenuniv.nl

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.

Show MeSH
Related in: MedlinePlus