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

PurposeThe 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.MethodsIgG 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.ResultsContrarily 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.ConclusionFluorescent molecular rotors are suitable probes to detect aggregation in polysorbate-containing IgG formulations.

[1]  H. Rehage,et al.  A new pyrene-based fluorescent probe for the determination of critical micelle concentrations. , 2007, The journal of physical chemistry. B.

[2]  Robert Gurny,et al.  A High Throughput Protein Formulation Platform: Case Study of Salmon Calcitonin , 2008, Pharmaceutical Research.

[3]  B. Kerwin.  Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways. , 2008, Journal of pharmaceutical sciences.

[4]  M. Hageman,et al.  Effects of buffer composition and processing conditions on aggregation of bovine IgG during freeze-drying. , 1999, Journal of pharmaceutical sciences.

[5]  M. Manning,et al.  Effect of Tween 20 on freeze-thawing- and agitation-induced aggregation of recombinant human factor XIII. , 1998, Journal of pharmaceutical sciences.

[6]  M. Lindgren,et al.  Detection and characterization of aggregates, prefibrillar amyloidogenic oligomers, and protofibrils using fluorescence spectroscopy. , 2005, Biophysical journal.

[7]  H. Cheung,et al.  A model for molecules with twisted intramolecular charge transfer characteristics: solvent polarity effect on the nonradiative rates of dyes in a series of water—ethanol mixed solvents , 1990 .

[8]  I. Oh,et al.  Fluorescence spectroscopy studies on micellization of poloxamer 407 solution , 2003, Archives of pharmacal research.

[9]  M. Haidekker,et al.  Effects of solvent polarity and solvent viscosity on the fluorescent properties of molecular rotors and related probes. , 2005, Bioorganic chemistry.

[10]  D. Kaplan,et al.  Emulsan quantitation by Nile red quenching fluorescence assay , 2005, Applied Microbiology and Biotechnology.

[11]  Xinmiao Fu,et al.  4,4'-Dianilino-1,1'-binaphthyl-5,5'-sulfonate, a novel molecule having chaperone-like activity. , 2005, Biochemical and biophysical research communications.

[12]  W. Akers,et al.  A molecular rotor as viscosity sensor in aqueous colloid solutions. , 2004, Journal of biomechanical engineering.

[13]  H. Burt,et al.  Determination of surfactant critical micelle concentration by a novel fluorescence depolarization technique. , 1996, Journal of biochemical and biophysical methods.

[14]  Randall J Mrsny,et al.  Formulation and delivery issues for monoclonal antibody therapeutics. , 2006, Advanced drug delivery reviews.

[15]  C E Kung,et al.  Fluorescent molecular rotors: a new class of probes for tubulin structure and assembly. , 1989, Biochemistry.

[16]  V. Bumelis,et al.  Congo red interaction with alpha-proteins. , 2006, Acta biochimica Polonica.

[17]  W. Jiskoot,et al.  Structural properties of monoclonal antibody aggregates induced by freeze-thawing and thermal stress. , 2009, European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences.

[18]  J A Frangos,et al.  New fluorescent probes for the measurement of cell membrane viscosity. , 2001, Chemistry & biology.

[19]  W. Vaz,et al.  Determination of the critical micelle concentration of surfactants using the fluorescent probe N-phenyl-1-naphthylamine. , 1986, Analytical biochemistry.

[20]  P. Guptasarma,et al.  Hydrophobic dye inhibits aggregation of molten carbonic anhydrase during thermal unfolding and refolding , 1999, Proteins.

[21]  Y. J. Wang,et al.  Dual effects of Tween 80 on protein stability. , 2008, International journal of pharmaceutics.

[22]  P. Acharya,et al.  Stability Studies on a Lipase from Bacillus subtilis in Guanidinium Chloride , 2003, Journal of protein chemistry.

[23]  Wim Jiskoot,et al.  Online fluorescent dye detection method for the characterization of immunoglobulin G aggregation by size exclusion chromatography and asymmetrical flow field flow fractionation. , 2008, Analytical biochemistry.

[24]  W. Margolin,et al.  Inhibition of Assembly of Bacterial Cell Division Protein FtsZ by the Hydrophobic Dye 5,5′-Bis-(8-anilino-1-naphthalenesulfonate)* , 1998, The Journal of Biological Chemistry.

[25]  G. Winter,et al.  The effects of Tween 20 and sucrose on the stability of anti-L-selectin during lyophilization and reconstitution. , 2001, Journal of pharmaceutical sciences.

[26]  K. Bhattacharyya,et al.  Non-radiative pathways of anilino-naphthalene sulphonates: twisted intramolecular charge transfer versus intersystem crossing , 1992 .

[27]  Theodore W Randolph,et al.  Silicone oil- and agitation-induced aggregation of a monoclonal antibody in aqueous solution. , 2009, Journal of pharmaceutical sciences.

[28]  W. Norde.  Adsorption of proteins at solid-liquid interfaces. , 1995 .

[29]  Etienne Weiss,et al.  Therapeutic antibodies: successes, limitations and hopes for the future , 2009, British journal of pharmacology.

[30]  Naoki Kamo,et al.  Oscillating frequency of piezoelectric quartz crystal in solutions , 1990 .

[31]  Wim Jiskoot,et al.  Extrinsic Fluorescent Dyes as Tools for Protein Characterization , 2008, Pharmaceutical Research.

[32]  Hanns-Christian Mahler,et al.  Shaken, not stirred: mechanical stress testing of an IgG1 antibody. , 2008, Journal of pharmaceutical sciences.

[33]  J. Philo.  Is any measurement method optimal for all aggregate sizes and types? , 2006, The AAPS Journal.