Use of Raman Microspectroscopy to Study Partioning of Molecules During Freeze-Processing of Protein Solutions

Biostabilization processes, based on desiccation, cooling or freezing, are surface-induced and thus are inevitably governed by scale-dependent kinetic constraints. During processing, these kinetic constraints induce heterogeneity (solute, and solvent concentration gradients, thermal gradients, and eventually, phase separation in the medium). During freezing, ice crystallization induces thermal (due to latent heat release), and concentration gradients (due to solute rejection) causing phase separation in the medium (i.e., formation of regions at different thermodynamic states). For example, during a typical cryopreservation application initially the bulk water freezes, increasing the concentration of the solutes in the rest of the unfrozen solution. Upon further cooling, the freeze-concentrated solution may also vitrify (or some constituents may crystallize), creating at least two distinct regions within the same medium with two distinct states.