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


CONCLUSIONS
Fluorescent molecular rotors are suitable probes to detect aggregation in polysorbate-containing IgG formulations.
Hawe
Andrea
A
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
Filipe
Vasco
V
Jiskoot
Wim
W
D000911:Antibodies, Monoclonal / Q000737:chemistry; D002626:Chemistry, Pharmaceutical; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D015335:Molecular Probes / Q000737:chemistry*; D011136:Polysorbates / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D015335:Molecular Probes; D011136:Polysorbates; D011994:Recombinant Proteins