Protein freeze concentration and micro-segregation analysed in a temperature-controlled freeze container

To examine effects of varied freezing conditions on the development of spatial heterogeneity in the frozen protein solution, macroscopic freeze concentration and micro-segregation of bovine serum albumin (BSA) were investigated in a temperature-controlled 200-ml freeze container. Freezing to -40 °C promoted formation of protein concentration gradients (69-114 μg ml-1) in frozen samples taken from 12 different freezer positions, whereby slow freezing in 4 h or longer facilitated the evolution of strong spatial heterogeneities and caused local concentration increases by 1.15-fold relative to the initial protein concentration (100 μg ml-1). To visualize protein micro-segregation during phase separation, BSA was conjugated with fluorescein isothiocyanate and confocal laser scanning fluorescence microscopy was used to localize and size the freeze-concentrated protein regions. Slow freezing resulted in distinctly fewer and larger protein domains in the frozen bulk than fast freezing. Surface stress on the protein during freezing would therefore be minimized at low cooling rates; microscopic freeze concentration would however be highest under these conditions, potentially favoring protein aggregation.