High Throughput Differential Scanning Fluorimetry (DSF) Formulation Screening with Complementary Dyes to Assess Protein Unfolding and Aggregation in Presence of Surfactants
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 Wordsaggregation 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
Dynamic light scattering
Differential scanning fluorimetry
Intrinsic protein fluorescence
Merck research laboratories
Real time polymerase chain reaction
Aggregation transition temperature of CRM197
Melting (unfolding) transition temperature of CRM197
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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