Abstract
Purpose
The purpose was to evaluate DSF for high throughput screening of protein thermal stability (unfolding/ aggregation) across a wide range of formulations. Particular focus was exploring PROTEOSTAT® – a commercially available fluorescent rotor dye – for detection of aggregation in surfactant containing formulations. Commonly used hydrophobic dyes (e.g. SYPRO™ Orange) interact with surfactants, complicating DSF measurements.
Methods
CRM197 formulations were prepared and analyzed in standard 96-well plate rT-PCR system, using SYPRO™ Orange and PROTEOSTAT® dyes. Orthogonal techniques (DLS and IPF) are employed to confirm unfolding/aggregation in selected formulations. Selected formulations are subjected to non-thermal stresses (stirring and shaking) in plate based format to characterize aggregation with PROTEOSTAT®.
Results
Agreement is observed between SYPRO™ Orange (unfolding) and PROTEOSTAT® (aggregation) DSF melt temperatures across wide range of non-surfactant formulations. PROTEOSTAT® can clearly detect temperature induced aggregation in low concentration (0.2 mg/mL) CRM197 formulations containing surfactant. PROTEOSTAT® can be used to explore aggregation due to non-thermal stresses in plate based format amenable to high throughput screening.
Conclusions
DSF measurements with complementary extrinsic dyes (PROTEOSTAT®, SYPRO™ Orange) are suitable for high throughput screening of antigen thermal stability, across a wide range of relevant formulation conditions – including surfactants –with standard, plate based rT-PCR instrumentation.
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Abbreviations
- λem :
-
Fluorescence emission wavelength
- λex :
-
Fluorescence excitation wavelength
- λweighted :
-
Weighted average (tryptophan) emission wavelength
- ANS:
-
8-Anilinonaphthalene-1-sulfonic acid fluorescent dye
- CCVJ:
-
9-(2-carboxy-2-cyanovinyl)julolidine fluorescent rotor dye
- CRM197:
-
Modified diphtheria toxin (CRM197)
- DCVJ:
-
9-Julolidinylmethylenemalononitrile fluorescent rotor dye
- dH,min :
-
Hydrodynamic radius
- DLS:
-
Dynamic light scattering
- DSF:
-
Differential scanning fluorimetry
- HT:
-
High throughput
- IPF:
-
Intrinsic protein fluorescence
- mAb:
-
Monoclonal antibody
- MRL:
-
Merck research laboratories
- rT-PCR:
-
Real time polymerase chain reaction
- Tagg :
-
Aggregation transition temperature of CRM197
- Tm :
-
Melting (unfolding) transition temperature of CRM197
References
Capelle MAH, Gurny R, Arvinte T. High throughput screening of protein formulation stability: practical considerations. Eur J Pharm Biopharm. 2007;65(2):131–48.
Lavinder JJ, Hari SB, Sullivan BJ, Magliery TJ. High-throughput thermal scanning: a general, rapid dye-binding thermal shift screen for protein engineering. J Am Chem Soc. 2009;131(11):3794–5.
He F, Woods CE, Becker GW, Narhi LO, Razinkov VI. High-throughput assessment of thermal and colloidal stability parameters for monoclonal antibody formulations. J Pharm Sci. 100(12):5126–41.
Niesen FH, Berglund H, Vedadi M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc. 2007;2(9):2212–21.
He F, Hogan S, Latypov RF, Narhi LO, Razinkov VI. High throughput thermostability screening of monoclonal antibody formulations. J Pharm Sci. 2010;99(4):1707–20.
He F, Raznikov VI, Middaugh CR, Becker GW. High-throughput biophysical approaches to therapeutic protein development. In: Nahri LO, editor Biophysics for therapeutic protein development Springer Science; 2013.
Nashine VC, Kroetsch AM, Sahin E, Zhou R, Adams ML. Orthogonal high-throughput thermal scanning method for rank ordering protein formulations. AAPS PharmSciTech. 2013;14(4):1360–6.
Bhambhani A, Kissmann JM, Joshi SB, Volkin DB, Kashi RS, Russell Middaugh C. Formulation design and high-throughput excipient selection based on structural integrity and conformational stability of dilute and highly concentrated IgG1 monoclonal antibody solutions. J Pharm Sci. 2012;101(3):1120–35.
Morefield GL. A rational, systematic approach for the development of vaccine formulations. AAPS J. 2011;13(2):191–200.
Li Y, Mach H, Blue JT. High throughput formulation screening for global aggregation behaviors of three monoclonal antibodies. J Pharm Sci. 2011;100(6):2120–35.
Shi S, Semple A, Cheung J, Shameem M. DSF method optimization and its application in predicting protein thermal aggregation kinetics. J Pharm Sci. 2013;102(8):2471–83.
Menzen T, Friess W. High-throughput melting-temperature analysis of a monoclonal antibody by differential scanning fluorimetry in the presence of surfactants. J Pharm Sci. 2013;102(2):415–28.
Hawe A, Filipe V, Jiskoot W. Fluorescent molecular rotors as dyes to characterize Polysorbate-containing IgG formulations. Pharm Res. 2010;27(2):314–26.
Haidekker MA, Brady TP, Lichlyter D, Theodorakis EA. Effects of solvent polarity and solvent viscosity on the fluorescent properties of molecular rotors and related probes. Bioorg Chem. 2005;33(6):415–25.
Hawe A, Sutter M, Jiskoot W. Extrinsic fluorescent dyes as tools for protein characterization. Pharm Res. 2008;25(7):1487–99.
Ablinger E, Leitgeb S, Zimmer A. (2013) Differential scanning fluorescence approach using a fluorescent molecular rotor to detect thermostability of proteins in surfactant-containing formulations. Int J Pharm. 2013;441 (1-2):255–260. https://doi.org/10.1016/j.ijpharm.2012.11.035.
Kayser V, Chennamsetty N, Voynov V, Helk B, Trout BL. Conformational stability and aggregation of therapeutic monoclonal antibodies studied with ANS and thioflavin T binding. MAbs. 2011;3(4):408–11.
Stsiapura VI, Maskevich AA, Kuzmitsky VA, Uversky VN, Kuznetsova IM, Turoverov KK. Thioflavin T as a molecular rotor: fluorescent properties of Thioflavin T in solvents with different viscosity. J Phys Chem B. 2008;112(49):15893–902.
Krebs MRH, Bromley EHC, Donald AM. The binding of thioflavin-T to amyloid fibrils: localisation and implications. J Struct Biol. 2005;149(1):30–7.
Naiki H, Higuchi K, Hosokawa M, Takeda T. Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavine T. Anal Biochem. 1989;177(2):244–9.
Maskevich AA, Stsiapura VI, Kuzmitsky VA, Kuznetsova IM, Povarova OI, Uversky VN, et al. Spectral properties of Thioflavin T in solvents with different dielectric properties and in a fibril-incorporated form. J Proteome Res. 2007;6(4):1392–401.
PROTEOSTAT® Protein aggregation assay: for microplates or flow cytometry: product manual [internet]. [cited April 6, 2017]. Available from: http://www.enzolifesciences.com.
Malito E, Bursulaya B, Chen C, Surdo PL, Picchianti M, Balducci E, et al. Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197. Proc Natl Acad Sci. 2012;109(14):5229–34.
Crane DT, Bolgiano B, Jones C. Comparison of the diphtheria mutant toxin, Crm197, with a Haemophilus Influenzae type-b polysaccharide-Crm197 conjugate by optical spectroscopy. Eur J Biochem. 1997;246(2):320–7.
Prevention CfDCa. Vaccine excipient & media summary. Epidemiology and prevention of vaccine-preventable diseases, 13th Edition, 2015.
Lee LHB, Milan S. Effect of increased CRM197 carrier protein dose on meningococcal C bactericidal antibody response. Clin Vaccine Immunol. 2012;19(4):551–6.
Roberts CJ. Therapuetic protein aggregation: mechanisms, design, and control. Trends Biotechnol. 2014;32(7):372–80.
Patist A, Bhagwat SS, Penfield KW, Aikens P, Shah DO. On the measurement of critical micelle concentrations of pure and technical-grade nonionic surfactants. J Surfactant Deterg. 2000;3(1):53–8.
Batrakova EV, Han H-Y, Alakhov VY, Miller DW, Kabanov AV. Effects of Pluronic Block Copolymers on Drug Absorption in Caco-2 Cell Monolayers. 1998;15(6):850–5.
Ćirin DM, Poša MM, Krstonošić VS. Interactions between sodium cholate or sodium Deoxycholate and nonionic surfactant (tween 20 or tween 60) in aqueous solution. Ind Eng Chem Res 51(9):3670–6.
ACKNOWLEDGMENTS AND DISCLOSURES
The authors gratefully acknowledge Henryk Mach for guidance with intrinsic protein fluorescence measurements and useful discussions. We gratefully acknowledge Brian K. Meyer and Christopher L. Daniels for reviewing the manuscript. We also thank MRL Vaccine Bioprocess for supplying CRM197 for this study. S. M. McClure, P. L. Ahl, and J.T. Blue are employees of Merck Sharp & Dohme Corp.
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Figure S1
Additional results from Fig. 1 in text. (a). Tm vs. pH, by NaCl [mM] from 96 well pH-NaCl screen of CRM197 using SYPRO™ Orange dye (unfolding)). (b). Tagg vs. pH, by NaCl [mM] from 96 well pH-NaCl screen of CRM197 using PROTEOSTAT® dye (aggregation)). (c). Tagg vs. Tm pH-NaCl screen data, where linear least square fit line is Tagg = 0.97Tm + 3.46°C (R2 = 0.93). Note: Tm and Tagg values for pH = 4.5 (no transitions observed) and control (no pH buffer) are not included. (GIF 179 kb)
Figure S2
SYPRO™ Orange Tm results from CRM197 excipient screen (excipient, by concentration). Bars colored by excipient type. Error bars represent standard deviation of duplicate measurements. Red line in figure is Tm for the base formulation (30 mM HEPES, pH = 7.5, 0 mM NaCl, no excipient) for reference. (GIF 284 kb)
Figure S3
PROTEOSTAT® Tagg results from CRM197 excipient screen (excipient, by concentration). Bars colored by excipient type. Error bars represent standard deviation of duplicate measurements. Red line in figure is Tagg for the base formulation (30 mM HEPES, pH = 7.5, 0 mM NaCl) for reference. (GIF 314 kb)
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McClure, S.M., Ahl, P.L. & Blue, J.T. High Throughput Differential Scanning Fluorimetry (DSF) Formulation Screening with Complementary Dyes to Assess Protein Unfolding and Aggregation in Presence of Surfactants. Pharm Res 35, 81 (2018). https://doi.org/10.1007/s11095-018-2361-1
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DOI: https://doi.org/10.1007/s11095-018-2361-1