How to choose between Differential Scanning Fluorimetery (DSF) vs Differential Scanning Calorimetry (DSC) in biopharmaceutical research
Protein stability is a critical parameter in biopharmaceutical research. In recent years, Differential Scanning Fluorimetry (DSF) was added to biophysical toolboxes of pharmaceutical companies and used for characterization of protein unfolding. However, with increased numbers of the new modalities of drugs entering development pipelines comes the realization of the lack of versatility of DSF technology. As pharmaceutical research is increasingly prioritizing biomolecules, such as antibody drug conjugates, bispecifics, nucleotides and mRNA lipid nanoparticles, DSF is falling behind.
Enter Differential Scanning Calorimetry (DSC). DSC is a multimodal technique with a wide range of applications beyond measuring protein thermal stability. What’s more, it provides a more complete picture than DSF when measuring protein thermostability, which can improve decision-making and reduce the likelihood of false positives.
How does Differential Scanning Fluorimetry (DSF) work?
Differential Scanning Fluorimetry (DSF) is a fluorescence-based method that measures protein stability by detecting changes in fluorescence as a protein unfolds. As temperature increases, a fluorescent dye “tags” exposed hydrophobic regions of the protein, generating a signal indicative of unfolding events.
This means that DSF is limited to proteins with intrinsic fluorophores or those that can be tagged. Co-solutes, including the dye added to the sample, can also interfere with fluorescence signals, reducing reliability. And it only detects unfolding events – it doesn’t provide data on other thermodynamic parameters, such as enthalpy changes.
What are the advantages of Differential Scanning Calorimetry?
Differential Scanning Calorimetry (DSC) directly measures the heat absorbed or released during protein unfolding without the need for fluorescent dyes. It provides a complete thermodynamic profile that includes the unfolding temperature (Tm) – the only parameter provided by DSF – as well as enthalpy changes (ΔH) and heat capacity (Cp).
The removal of fluorescent dyes makes DSC applicable to proteins, nucleic acids, lipids, and their assemblies. In addition, because DSC does not rely on optical, signal-based measurements, it is free from artifacts caused by quenching, protein aggregation, or light scattering.
The repeatability and reliability of DSF is unmatched by DSC, which is a more versatile technique. As a result, DSC instruments are a highly cost-effective and future-proof investment.
DSF vs. DSC for protein stability measurements
DSF offers the advantage of high-throughput screening and requires smaller sample volumes, making it a practical choice for early-stage studies where speed and efficiency are a priority. However, it is limited by its reliance on fluorescence-based measurements, which can introduce variability and limit its applicability.
In contrast, DSC provides comprehensive and highly reproducible thermodynamic data, making it essential for biologics formulation development, biosimilarity assessment, and regulatory submissions. It is not affected by optical artifacts and can be used with a wide range of buffers and co-solvents, ensuring consistent and reliable results.
| Feature | DSC | DSF |
| Label-free analysis | ✔ | X |
| Applicable to proteins, nucleic acids, lipids | ✔ | X |
| High-throughput screening | X | ✔ |
| Detailed thermodynamic data | ✔ | X |
| Sensitive to all levels of protein structure | ✔ | X |
| Unaffected by optical artifacts | ✔ | X |
| Low sample volume requirement | X | ✔ |
| Highly reproducible | ✔ | X |
| Minimal buffer interference | ✔ | X |
Ultimately, the choice between DSF and DSC depends on the specific requirements of the application. If rapid screening and minimal sample consumption are required, DSF may be the preferred technique. However, if accurate, reproducible, and detailed thermodynamic insight is required, DSC remains the superior method.
For biopharmaceutical development, where stability characterization influences formulation decisions and regulatory approval, DSC remains the gold standard. With its precision and reliability, MicroCal PEAQ-DSC is an unparalleled tool for researchers seeking the most comprehensive approach to protein stability analysis.
If you have any questions about DSC or would like a demo of the MicroCal PEAQ-DSC, please contact our experts today.
Further reading
- Dive into our 3-part blog series on the role DSC can play in speeding up vaccine candidate selection
- Watch the webinar: Differential scanning calorimetry: Robust and powerful physical characterization of therapeutic protein products