Insights into protein-polysorbate interactions analysed by means of isothermal titration and differential scanning calorimetry.

Therapeutic proteins formulated as liquid solutions at high protein concentration are very sensitive to chemical and physical degradation. Especially avoiding the formation of protein aggregates is very crucial for product quality. In order to stabilize the colloidal properties of protein therapeutics various excipient are used. Especially the detergents polysorbate 20 and 80 are common. However, the mechanism upon which the detergents protect the protein from aggregation is not really known. The present study investigates the interaction of polysorbate 20 and 80 with different proteins: lysozyme, bovine serum albumin (BSA) and an immunoglobulin. The interaction and binding of the detergents to the proteins is investigated by isothermal titration calorimetry (ITC). From ITC the thermodynamic parameters (DeltaH: change in enthalpy, DeltaS: entropy and DeltaG: free energy) upon binding are derived as well as the binding constant K (a). The thermal stability of the proteins in the presence of the detergent is assessed by differential scanning calorimetry (DSC). The results show that both detergents bind to BSA with K (a) between 8 and 12 x 10(3) M(-1) with DeltaH -50 to -60 kJ/mol (25 degrees C). One to two detergent molecules bind to BSA. The presence of both detergents induces a weak stabilisation of the thermal denaturation properties of BSA. However, the interaction of polysorbate 20 and 80 with lysozyme and the immunoglobulin is quite negligible. The presence of the detergents up to a concentration of 2 mM has no impact on the heat capacity curve neither a destabilisation nor a stabilisation of the native conformation is observed.