Fluorescent dyes as tools to detect changes in higher order. protein structures

Transcription

1 Fluorescent dyes as tools to detect changes in higher order Wim Jiskoot protein structures native stressed protein + bound dye protein native intermediate unfolded unbound dye partially unfolded aggregate disordered aggregate Division of Drug Delivery Technology Leiden/Amsterdam Center for Drug Research (LACDR) Barcelona 22 March 2011

2 Protein aggregation: an immunological concern Simplified summary: protein aggregates are immunogenic

3 Protein aggregation: an analytical challenge Frokjaer & Otzen, Nat Rev Drug Disc 4 (2005),

4 Range of detection of various analytical techniques Visual inspection Microscope Light Obscuration Micro Flow Imaging Field Flow AF4 Fractionation Nanoparticle Tracking Analysis Dynamic Light Scattering SDS-PAGE SE-HPLC 1 nm 10 nm 100 nm 1 µm 10 µm 100 µm 1 mm Protein monomer Soluble aggregates Insoluble aggregates Courtesy of Michael Wiggenhorn, Coriolis

5 Protein aggregation: an analytical challenge Look at aggregates from different angles! Dynamic light scattering hydrodynamic size Analytical ultracentrifugation soluble and insoluble aggregates Extrinsic fluorescent dyes structural changes aggregates UV/VIS presence of particles structural changes Multi-angle laser light scattering determination MW HP-SEC soluble aggregates FTIR intermolecular β-sheets Turbidimetry, light obscuration presence of particles SDS-PAGE covalent aggregates Emerging techniques MFI, MacroIMS, NanoSight, Asymmetrical flow-field flow fractionation soluble and insoluble aggregates

6 Fluorescent dyes for monitoring protein aggregation Addition of dye Detection of few aggregates, in presence of excess native protein No limitations with respect to aggregate size Well suited to use in high-throughput assay formats Can be used in combination with separation methods

7 Fluorescent dyes ANS Nile red Bis-ANS Thioflavin T DCVJ Congo red Hawe et al. (2008) Pharm. Res. 25:

8 Bis ANS fluorescence with heat stressed IgG 1.0 mg/ml IgG in 100 mm phosphate ph µm Bis ANS 3.5E+05 fluorescence intensity [a.u.] 3.0E E E E E E C 75 C 70 C unstressed 0.0E emission [nm] Hawe et al. (2008) Analytical Biochemistry 378:

9 5 µm Bis ANS with increasing concentrations of heatstressed IgG Applicable to a wide protein concentration range (<0.05 mg/ml to > 100 mg/ml)

10 Enbrel 50 mg/ml pre filled syringe, 7 days 50 C ns = not significant, * p<0.05, ** p<0.01, *** p<0.001 day 1 day 2 day 3 day 4 day 7 Bis ANS *** *** *** *** *** DLS, Zave ns ns *** *** *** DLS, PDI ns ns *** *** * HP SEC ns ** *** *** *** LO, >1 µm ns ns ns ns ns LO, >10 µm ns ns ns ns ns LO, >25 µm ns ns ** ns ns CD 222 nm * ns *** *** *** UV 2 nd der ns ns ns ns *** ELISA ns ns ns * *** Van Maarschalkerweerd et al. (2011) Eur. J. Pharm. Biopharm., in press

11 Combination of Bis ANS with size exclusion exclusion HPLC 1 µm Bis-ANS in mobile phase Size-exclusion HPLC Fluorescence detection Excitation 385 nm, Emission 488 nm

12 Size exclusion HPLC: comparing UV and Bis ANS detection UV detection Bis ANS detection 0.03 relative UV signal C 70 C before relative fluorescence signal C 70 C before time [min] time [min] Hawe et al. (2008) Analytical Biochemistry 378:

13 Size exclusion HPLC: comparing UV, Bis ANS and MALLS detection 1.0 mg/ml IgG unstressed and stressed at 80 C Bis-ANS UV MALLS relative detector signals MW [kda] time [min] Hawe et al. (2008) Analytical Biochemistry 378:

14 Do the dyes influence aggregation behavior? Dyes can either promote or inhibit aggregation (as any substance used in analysis, HP SEC, ) Pharm. Res. 25: (2008) In our hands, effects are negligible when the dye is added after applying stress, just before analysis However, always to be tested (HP SEC, DLS )

15 Do the dyes influence aggregation behavior? HP SEC of heat stressed IgG UV280 absorbance in mobile phase without dye, with Bis ANS or CCVJ 150 UV signal 280 nm no dye Bis-ANS CCVJ time [min] no dye Bis-ANS CCVJ time [min] Hawe et al. (2009) Eur. J. Pharm. Sci. 38: 79 87

16 Do all aggregates interact with the dyes? Heat stressed versus freeze thawed IgG HP SEC with UV280 absorption detection 5.0E E E E E-02 unstressed FT 10 min 75 C UV [mau] 5.9E E E E E E E time [min] Hawe et al. (2009) Eur. J. Pharm. Sci. 38: 79 87

17 Do all aggregates interact with the dyes? Heat stressed versus freeze thawed IgG HP SEC with Bis ANS fluorescence detection 1.2E+00 Bis-ANS fluorescence [mau] 9.9E E E E E-01 unstressed FT 10 min 75 C -1.0E time [min] Hawe et al. (2009) Eur. J. Pharm. Sci. 38: 79 87

18 Do all aggregates interact with the dyes? Second derivative FTIR spectrum of isolated aggregates 0.1 second derivative xFT, prec. 77 C heat, prec. NS wavenumber [cm -1 ] Hawe et al. (2009) Eur. J. Pharm. Sci. 38: 79 87

19 Do surfactants interfere with dye detection? Almost 70% of the marketed monoclonal antibody formulations contain polysorbate 20 or polysorbate 80 Concentrations range between 0.001% (w/v) polysorbate 80 (Reopro ) and 0.16% (w/v) polysorbate 20 (Raptiva ), with most formulations containing between % polysorbate 20 or 80 Chames et al. (2009) Therapeutic antibodies: successes, limitations and hopes for the future. Br J Pharmacol 157: Daugherty & Mrsny (2006) Formulation and delivery issues for monoclonal antibody therapeutics. Adv Drug Deliv Rev 58: FDA ReoPro@, Abciximab, For intravenous administration, (BLA) EMEA European Public Assessment Report of Raptiva (25/11/2008 Raptiva H C 542 II 26) EMEA website

20 Bis ANS and polysorbate Bis ANS fluorescence as function of polysorbate 20 concentration in 100 mm phosphate, ph 7.2, containing 1.0 mg/ml IgG intensity of emission maximum [a.u.] HT+PS20 NS+PS20 placebo+ps polysorbate 20 concentration [%] Hawe et al. (2010) Pharm. Res. 27:

21 Fluorescent molecular rotors DCVJ CCVJ 9-(dicyanovinyl)-julolidine 9-(2-carboxy-2-cyanovinyl)- julolidine Free rotation possible in excited state Deviation from planar structure abolishes fluorescence Rotation hindered in viscous environments

22 Bis ANS fluorescence versus CCJV fluorescence Bis-ANS fluorescence is sensitive to polarity CCVJ fluorescence is sensitive to viscosity Hawe et al. (2010) Pharm. Res. 27:

23 CCVJ and polysorbate CCVJ fluorescence as function of polysorbate 20 concentration in 100 mm phosphate, ph 7.2, containing 1.0 mg/ml IgG intensity of emission maximum [a.u.] HT+PS20 NS+PS20 placebo+ps polysorbate 20 concentration [%] Hawe et al. (2010) Pharm. Res. 27:

24 Use of CCVJ to analyze Humira 40 mg (contains 0.1% polysorbate 80) 5 μm CCVJ (Exc: 435 nm) fluorescence intensity [a.u.] min 65 C 10 min 60 C NS placebo emission wavelength [nm] Hawe et al. (2010) Pharm. Res. 27:

25 Conclusions Fluorescent dyes are highly sensitive, selective and versatile tools to detect structurally altered protein Fluorescent dye based assays complement our analytical arsenal to monitor protein aggregation Not all aggregates interact strongly with the dyes, so no dye signal does not mean no aggregates Molecular rotors such as DCVJ and CCVJ can be used for polysorbate containing formulations

26 Protein aggregation: further reading Pharmaceutical Research, online first

27 Acknowledgments The ProFormA team Miranda van Beers Vasco Filipe Riccardo Torosantucci Andrea Hawe Robert Poole Ruediporn (Sun) Tantipolphan Basak Kukrer UIPS Utrecht Institute for Pharmaceutical Sciences Marc Sutter Tudor Arvinte And may others students, technicians, collaborators

28 Thank you!! Reversible water aggregates adsorbed on a bicycle Wim Jiskoot

29 Fluorescent dyes physical principle absorption emission nonradiative decay S 2 S 1 solvent relaxation S 1 (T)ICT S (T)ICT S 0 hydrophobic environment; aggregated protein High fluorescence hydrophilic environment; native protein Low fluorescence; red shift Hawe et al. (2008) Pharm. Res. 25:

30 Fluorescent dyes physical principle ground state strong fluorescence, blue shift twisted intramolecular charge transfer (TICT): low fluorescence, red shift Hawe et al. (2008) Pharm. Res. 25:

31 Use of multiple dyes provides additional info Insulin: amorphous aggregates versus fibrils Relative intensity [a.u] Bis-ANS emission [nm] Relative intensity [a.u] Thioflavin T emission [nm] native amorphous aggregates fibrils Tantipolphan, unpublished results

32 Fluorescent dyes for monitoring protein aggregation native protein stressed protein + bound dye native intermediate unfolded unbound dye emission [nm] partially unfolded aggregate disordered aggregate Examples: Nile Red, ANS, Bis-ANS, Thioflavin T, DCVJ,

33 Bis ANS fluorescence is sensitive to polarity ethanol fluorescence intensity [a.u.] Decreasing polarity methanol glycerol buffer wavelength [nm] Hawe et al. (2010) Pharm. Res. 27:

34 CCVJ fluorescence is sensitive to viscosity 8000 η(mpa.s; 25 ) fluorescence intensity [a.u.] glycerol ethanol methanol buffer wavelength [nm] Hawe et al. (2010) Pharm. Res. 27: