Additives affecting thermal stability of salmon calcitonin in aqueous solution and structural similarity in lyophilized solid form

The effects of additives on conformational stability and structural similarity of salmon calcitonin (sCT) in aqueous solution and lyophilized solid form were investigated by using transmission Fourier transform infrared (FT-IR) microspectroscopy with second-derivative spectral analysis. To compare the structural similarity between native sCT and lyophilized sCT samples with or without additive, the spectral correlation coefficient analysis between two second-derivative IR spectra was applied. Two types of additives were also selected: sugars/polols [trehalose, sucrose, mannitol and 2-hydroxypropyl-β-cyclodextrin (HP-βCD)] and polymers [dextran, heparin and human serum albumin (HSA)]. When trehalose, mannitol or dextran was respectively co-incubated with sCT in 40 °C aqueous solution, these additives slightly changed the secondary conformation of sCT. However, sucrose or HP-βCD accelerated the intramolecular β-sheet formation of sCT due to the appearance of IR peak at 1631 cm−1. A precipitation was occurred in the sCT aqueous solution after co-incubation with heparin or HSA, in which the ionic interaction and/or hydrophobic adsorption might be responsible for this result. A strong peak at 1630 cm−1 (intramolecular β-sheet) with a minor peak at 1618 cm−1 (intermolecular β-sheet) was also evidenced in the second-derivative IR spectrum of the sCT precipitate. The secondary conformation of native sCT after lyophilization was markedly altered from the predominant α-helix to random coil/β-sheet structures, but trehalose, mannitol or sucrose had the greatest stabilization effect on the lyophilized sCT due to a higher structural similarity. The secondary structure of sCT was perturbed during co-lyophilization with HP-βCD, dextran, heparin or HSA. The result of this study indicates that both mannitol and trehalose would serve as optimal stabilizers for sCT in both aqueous solution and lyophilized formulation.