Role of Poloxamer 188 in Preventing Ice-Surface-Induced Protein Destabilization during Freeze-Thawing

The phase behavior of poloxamer 188 (P188) in aqueoussolutions,characterized by differential scanning calorimetry (DSC) and synchrotronX-ray diffractometry, revealed solute crystallization during both freezing and thawing. Sucrose and trehalose inhibited P188 crystallizationduring freeze-thawing (FT). While trehalose inhibited P188crystallization only during cooling, sucrose completely suppressedP188 crystallization during both cooling and heating. Lactate dehydrogenase(LDH) served as a model protein to evaluate the stabilizing effectof P188. The ability of P188, over a concentration range of 0.003-0.800%w/v, to prevent LDH (10 & mu;g/mL) destabilization was evaluated.After five FT cycles, the aggregation behavior (by dynamic light scattering)and activity recovery were evaluated. While LDH alone was sensitiveto interfacial stress, P188 at concentrations of & GE;0.100% w/vstabilized the protein. However, as the surfactant concentration decreased, protein aggregation after FT increased. The addition of sugar (1.0%w/v; sucrose or trehalose) improved the stabilizing function of P188at lower concentrations (& LE;0.010% w/v), possibly due to theinhibition of surfactant crystallization. Based on a comparison withthe stabilization effect of polysorbate (both 20 and 80), it was evidentthat P188 could be a promising alternative surfactant in frozen proteinformulations. However, when the surfactant concentration is low, thepotential for P188 crystallization and the consequent compromise inits functionality warrant careful consideration.