Protein Structural Stability by Nano-Differential Scanning Calorimetry

More than half of current protein therapeutics were developed in liquid dosage forms. However, conformational stability is a key issue for bioactivity of protein formulations [1]. Therefore, studies to understand the effect of pH, ionic strength and stabilizing agents on the structural stability of proteins in their formulation media can provide key insights for suitable formulation screen.

Nano-DSC is an important tool for studying the conformational stability of proteins in solution to support formulation development. Thermodynamic properties of proteins are evaluated by the enthalpy (∆H) of unfolding and the melting temperature Tm at which half the molecules are unfolded.

Conformational stability

Lysozyme has been used as a model protein to study the impact of buffer, pH, and sugar on the conformational stability. Lysozyme exhibited a shift in transition temperatures (Tm) in different buffers with varying pH values (Figure 1). Lower Tm (~61 °C) at pH 2 (glycine/HCl) and higher Tm at pH 5 (citrate buffer) was observed. Electrostatic interactions could have contributed partially to the stability of the native state.

In the presence of sucrose, the thermal transition temperature (ΔTm) increased with increasing sucrose concentration (Figure 2), which indicates that the protein has a more stable structure in sucrose. One hypothesis for the mechanism of structural stabilization of protein by sugar is the preferential hydration of proteins [2-4]. The preferential hydration mechanism proposes that sugar does not bind to proteins, and it is preferentially excluded from the vicinity of the protein. Therefore, water concentration sounding the protein is higher than in the bulk phase. The surface tension of water surrounding the protein increases due to the addition of sugars.

Domain structure of protein

Immunoglobulin molecules consist of two light chains and two heavy chains (Figure 3). The chains are linked by covalent interchain disulfide bonds and non-covalent interactions. The DSC thermogram of antibody IgG from rabbit serum exhibited a single transition peak with Tm at 78 °C (Figure 4). The denaturation occurred in a narrow temperature range, indicating the unfolding is highly cooperative. Upon reduction with dithiothreitol (DTT), the intra- and inter-chain disulfide bridges were cleaved by the reducing agents, resulting in unfolded or partially unfolded heavy and light chains. The DSC thermogram displayed several broad overlapping peaks, indicating independent unfolding of the subunits or cooperative domains over different temperature ranges (Figure 5).


  1. Carpenter, J. F., Chang, B. S., Garzon-Rodriguez, W., and Randolph, T. W. 2002. In “Rationale Design of stable protein formulations-theory and practice” (J. F. Carpenter and M.C. Manning eds.) Kluwer Academic/Plenum publishers, New York, pp. 109-133.
  2. Kaushik, J. K.; Bhat, R. Biol. Chem.. 2003, 278, 26458−26465.
  3. Kaushik, J. K.; Bhat, R. Phys. Chem. B. 1998, 102, 7058−7066.
  4. Timasheff, S. N. Natl. Acad. Sci. U. S. A., 2002, 99, 9721-9726.
  5. Harris, S. B. Larson, K. W. Hasel, A. McPherson. Biochemistry 1997, 36, 1581-1597.