Pharmaceutical Research

, 35:81 | Cite as

High Throughput Differential Scanning Fluorimetry (DSF) Formulation Screening with Complementary Dyes to Assess Protein Unfolding and Aggregation in Presence of Surfactants

  • Sean M. McClure
  • Patrick L. Ahl
  • Jeffrey T. Blue
Research Paper



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.


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®.


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.


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.

Key Words

aggregation fluorescence spectroscopy high-throughput screening modified diphtheria toxin (CRM197) vaccine formulation 



Fluorescence emission wavelength


Fluorescence excitation wavelength


Weighted average (tryptophan) emission wavelength


8-Anilinonaphthalene-1-sulfonic acid fluorescent dye


9-(2-carboxy-2-cyanovinyl)julolidine fluorescent rotor dye


Modified diphtheria toxin (CRM197)


9-Julolidinylmethylenemalononitrile fluorescent rotor dye


Hydrodynamic radius


Dynamic light scattering


Differential scanning fluorimetry


High throughput


Intrinsic protein fluorescence


Monoclonal antibody


Merck research laboratories


Real time polymerase chain reaction


Aggregation transition temperature of CRM197


Melting (unfolding) transition temperature of CRM197



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.

Supplementary material

11095_2018_2361_Fig7_ESM.gif (180 kb)
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)

11095_2018_2361_MOESM1_ESM.tif (106 kb)
High resolution image (TIFF 105 kb)
11095_2018_2361_Fig8_ESM.gif (285 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)

11095_2018_2361_MOESM2_ESM.tif (321 kb)
High resolution image (TIFF 321 kb)
11095_2018_2361_Fig9_ESM.gif (314 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)

11095_2018_2361_MOESM3_ESM.tif (371 kb)
High resolution image (TIFF 370 kb)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Sean M. McClure
    • 1
  • Patrick L. Ahl
    • 2
  • Jeffrey T. Blue
    • 2
  1. 1.Center for Materials Science and EngineeringMerck Sharp & Dohme CorpWest PointUSA
  2. 2.Vaccine Drug Product DevelopmentMerck Sharp & Dohme CorpWest PointUSA

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