Probing high-pressure stresses caused by ice-growth during freezing of biotherapeutics

Freezing can lead to irreversible damage on biotherapeutic macromolecules. This has been attributed to stresses like the ice interface, freeze-concentration and solute crystallisation. Although, our previous experiments suggest that freezing can also generate local high-pressure. We have observed additional X-ray diffraction peaks that cannot be assigned to hexagonal ice, while they matched with high-pressure forms; IceIII and IceIX. The expansion of crystallizing water, combined with physical restrictions form neighbouring ice crystals and increased viscosity of the freeze-concentrated solution may explain this effect. The objective of the proposed study is to confirm the additional X-ray diffraction peaks in frozen aqueous solutions, which showed some thermal diffuse scattering. Measurements will be performed at different temperatures, with pure water and solutions of proteins and low molecular weight solutes, aiming to clarify this critical variable that may have been overlooked.