The Role of Differential Scanning Fluorimetry (DSF) and Differential Scanning Calorimetry (DSC) in Protein Stability Assessment

Protein stability assessment is an extremely important parameter in biopharmaceutical research. In recent years, Differential Scanning Fluorimetry (DSF) has been widely used as a method to evaluate protein unfolding characteristics. DSF is relatively simple and allows for rapid screening from small sample sizes, making it useful in the early stages of research.

However, in the pharmaceutical development field, the emergence of various modalities such as antibody-drug conjugates, bispecific antibodies, nucleic acids, and mRNA lipid nanoparticles (LNPs) has increased the demand for more versatile measurement methods. In this trend, cases where the applicability of DSF is limited have also been observed.

On the other hand, Differential Scanning Calorimetry (DSC) is a versatile method with a wide range of applications beyond measuring thermal stability of proteins. Moreover, because it provides a more comprehensive picture when measuring protein thermal stability than DSF, it can lead to improved decision-making and reduced possibility of false positives.

What is Differential Scanning Fluorimetry (DSF)? Features and Points to Consider

DSF is a method that uses fluorescent dyes to capture changes in fluorescence associated with protein denaturation. As temperature increases, fluorescent dyes interact with the hydrophobic regions of proteins, detecting the progress of unfolding. This method is rapid and can be performed with small amounts of sample, offering advantages.

However, because it depends on fluorescent dyes, the reliability of the results can decrease for proteins that do not interact with the dye or samples easily affected by coexisting compounds. Additionally, DSF mainly measures T_m (melting temperature), without providing other thermodynamic information like enthalpy or heat capacity.

DSC Enables More Multifaceted Analysis

Differential Scanning Calorimetry (DSC) is a method that directly measures heat absorption or release associated with protein unfolding without the need for fluorescent dyes. With DSC, it is possible to obtain detailed thermodynamic profiles, including changes in T_m, enthalpy (ΔH), and heat capacity (C_p), allowing for deeper insights.

Moreover, because DSC is not influenced by optical artifacts and can be applied to various types of biomolecules (proteins, nucleic acids, lipids, etc.), it is increasingly recognized as a more reproducible and versatile measurement method.

Comparison and Utilization of DSF and DSC in Protein Stability Measurement

DSF is extremely useful for high-throughput screening and requires a small amount of sample, making it particularly advantageous in the early stages of screening where speed and efficiency are prioritized. Due to its simplicity, it is widely used as a method to support decision-making in the early research stages.

However, since it is based on fluorescence signals, variability in signals is likely to occur due to interactions with coexisting substances and dyes, and there are cases where application is difficult under certain sample conditions.

On the other hand, DSC provides more comprehensive and reproducible thermodynamic data, making it particularly effective in situations where accurate evaluation is required, such as in formulation development of biopharmaceuticals, biosimilar evaluations, and preparation of application materials to regulatory agencies. DSC is less affected by optical artifacts and maintains high reliability under various buffer or cosolvent conditions, so it can be confidently used for quality evaluation in later stages.

DSF and DSC each have their strengths, and it is important to utilize them according to research objectives. For instance, DSF is suitable for initial screenings and rapid evaluations of numerous samples. In contrast, DSC is an excellent choice for scenarios requiring more detailed and accurate stability assessments, such as formulation development, biosimilar evaluations, and preparation of submission documents to regulatory authorities.

In biopharmaceutical development, stability assessment is a crucial process directly linked to formulation design, quality control, and even regulatory approval. High-precision DSC instruments like MicroCal PEAQ-DSC provide reliable thermodynamic data and serve as extremely useful tools for researchers. Selecting the appropriate evaluation method according to the research stage and objectives contributes significantly to product development success.

MicroCal DSC Series for Biomolecule Stability Analysis in Solution

This device is capable of evaluating the thermal stability of biomolecules in solution with high sensitivity and reproducibility. It directly measures extremely small thermal changes associated with denaturation within biomolecules under temperature-controlled conditions.

This product is also available at academic pricing. Please contact us for more details.