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The purpose of this study was to investigate the effect of sucrose-glycine excipient systems on the stability of selected model proteins during lyophilization. Recovery of protein activity after freeze-drying was examined for the model proteins lactate dehydrogenase and glucose 6-phosphate dehydrogenase in a sucrose-glycine-based excipient system in which the formulation composition was system-atically varied. In a sucrose-only excipient system, activity recovery of both model proteins is about 80% and is independent of sucrose concentration over a range from 1 to 40 mg/mL. When both sucrose and glycine are used and the ratio of the 2 excipients is varied, however, activity recovery decreases in a pattern that is consistent with the inhibition of activity recovery by glycine crystals, despite the presence of an adequate amount of sucrose to afford protection. Annealing of sucrose-glycine formulations causes a small but significant decrease in activity recovery relative to unannealed controls, whereas no annealing effect is observed with sucrose-only formulations. Addition of 0.01% polysorbate 80 to the formulation resulted in complete recovery of activity, irrespective of the sucroseglycine ratio or annealing. Addition of the same concentration of polysorbate 80 to the reconstitution medium caused an increase in activity recovery for each formulation, but the overall pattern remained unchanged. The data are consistent with an interfacial model for lyophilization-associated loss of protein activity involving denaturation at a solid/freeze-concentrate interface.