Differential Scanning Calorimetry (DSC) is a technique used to characterize the stability of

a protein or other biomolecule directly in its native form. It does this by measuring the heat
change associated with the molecule’s thermal denaturation when heated at a constant
rate.

Measurement principle
A biomolecule in solution is in equilibrium between its native (folded) and denatured (unfolded)
conformations. The higher the thermal transition midpoint (Tm), the more stable the molecule.
DSC measures the enthalpy (∆H) of unfolding that results from heat-induced denaturation. It is
also used to determine the change in heat capacity (ΔCp) of denaturation. DSC can elucidate the
factors that contribute to the folding and stability of native biomolecules. These include
hydrophobic interactions, hydrogen bonding, conformational entropy and the physical
environment.

The precise and high quality data obtained from DSC provides vital information on protein stability
in process development, and in the formulation of potential therapeutic candidates.

Macromolecules and macromolecular assemblies (>5000 Daltons), such as proteins, nucleic

acids and lipids, can form well-defined structures that undergo thermally-induced conformational
changes. These structural rearrangements result in the absorption of heat caused by the
redistribution of non-covalent bonds. Differential scanning calorimeters measure this heat uptake.

How DSC works

The thermal core of a DSC system consists of two cells, a reference and a sample cell. The
device is designed to maintain the two cells at the same temperature, as they are heated.
Performing a measurement
To perform a DSC measurement, the reference cell is first filled with buffer and the sample cell
with the sample solution. These are then heated at a constant scan rate. The absorption of heat
that occurs when a protein unfolds causes a temperature difference (ΔT) between the cells,
resulting in a thermal gradient across the Peltier units. This sets up a voltage, which is converted
into power and is used control the Peltier to return ΔT (the temperature differential) to 0°C.
Alternatively, the cells can be allowed to reach thermal equilibrium passively through conduction.

Data generation and analysis

The enthalpy of protein unfolding is the area under the concentration-normalized DSC peak and
has units of calories (or joules) per mole. In certain cases, thermodynamic models can be fitted to
the data to obtain the Gibb's free energy (ΔG), the calorimetric enthalpy (ΔHcal), the van't Hoff
enthalpy (ΔHvH), the entropy (ΔS) and the change in the heat capacity (ΔCp) associated with the
transition.
 DSC is widely used in drug discovery and development. Key applications include:

 Characterization and selection of the most stable proteins or potential candidates in biotherapeutic
development
 Ligand interaction studies
 Rapid optimization of purification and manufacturing conditions
 Easy, rapid determination of optimum conditions for liquid formulations
 Quick stability indicating assay for target proteins to be used for screening
Related products
MicroCal DSC rangePowerful analytical tools for characterizing the stability of proteins

and other biomolecules

Measurement
Microcalorimetry, Label-free analysis
Temperature range
-10°C to 130°C
Sample throughput
2per 24h day to 50per 24h day
Technology
Differential Scanning Calorimetry