Observation of ice-solute interaction in freezing of trehalose and albumin solutions by using confocal Raman microscope equipped with directional solidification stage

Stability and activities of biopharmaceuticals after thawing largely depend on ice-solute interaction in freezing process. The aims of the present study were to observe microscopic distribution of protein or sugar that was trapped in the ice after freezing of their aqueous solutions and to evaluate protein denaturation. A confocal Raman microscope (CRM) combined with a directional solidification stage (DSS) was developed for this purpose. Trehalose and bovine serum albumin solutions at a concentration of 5 to 20 wt % were respectively enclosed in a microchannel. They were solidified on the DSS at different degree of supercooling, and then scanned by the CRM along the microchannel. The Raman imaging successfully exhibited variety in distribution of ice and solutes: uniform dispersion of the organic matters and ice crystals at highly supercooled area because of higher freezing speed, formation of solute-accumulated spots between ice crystals at lower supercooled area due to rejection of solutes, and relatively pure ice region near the ice front due to full rejection of solutes. It depended on the solute and its concentration, the degree of supercooling, and the freezing speed. An attempt to evaluate the freeze-induced denaturation of protein was also made with analyzing the Raman spectrum of α-helix band relative to that of CH band. An increase in denaturation was observed with repetitive freezing, which demonstrated the feasibility of denaturation detection. It however suggested that using the other type of protein that is more sensitive to freezing is preferable to evaluate site-dependent denaturation in-situ.