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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...

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Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
13:26

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Published on: September 13, 2014

DSF method optimization and its application in predicting protein thermal aggregation kinetics.

Shuai Shi1, Andrew Semple, Jason Cheung

  • 1Sterile Product Development Group, Bioprocess Development, Merck Sharp & Dohme Corp., Summit, New Jersey 07901, USA. shuai.shi@merck.com

Journal of Pharmaceutical Sciences
|June 12, 2013
PubMed
Summary
This summary is machine-generated.

Differential scanning fluorimetry (DSF) optimization is crucial for therapeutic protein development. Surfactants and dye concentrations impact protein thermal transitions and aggregation prediction, requiring careful method validation.

Keywords:
aggregation kineticsanalytical biochemistrybiopharmaceuticals characterizationbiotechnologydifferential scanning calorimetrydifferential scanning fluorimetryprotein aggregationprotein formulationsurfactant

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Area of Science:

  • Biochemistry
  • Protein Science
  • Analytical Chemistry

Background:

  • Differential scanning fluorimetry (DSF) is widely used in therapeutic protein development.
  • The influence of dyes and surfactants on protein structural transitions in DSF requires further investigation.

Purpose of the Study:

  • Optimize DSF methods for surfactant-containing formulations.
  • Benchmark DSF against differential scanning calorimetry (DSC).
  • Evaluate the predictive capability of thermal parameters for protein aggregation kinetics.

Main Methods:

  • Differential scanning fluorimetry (DSF) optimization with varying surfactant and dye concentrations.
  • Benchmarking DSF against differential scanning calorimetry (DSC).
  • Monitoring real-time thermal aggregation kinetics using size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC).

Main Results:

  • Surfactants generally necessitate medium-to-high protein concentrations for DSF.
  • High SYPRO® Orange concentration can lower protein thermal transitions.
  • DSF and DSC predict aggregation for MAb fragments, but not always for specific monoclonal antibodies (e.g., MAb3).
  • DSF results did not align with DSC or predict aggregation kinetics in a surfactant study.

Conclusions:

  • DSF method optimization is essential, particularly concerning surfactant and dye concentrations.
  • The predictive power of DSF/DSC for aggregation kinetics varies depending on the protein and formulation.
  • Further studies are needed to refine DSF applications in therapeutic protein development, especially in the presence of excipients.