Freeze-Drying of Proteins from a Sucrose-Glycine Excipient System: Effect of Formulation Composition on Initial Recovery of Protein Activity

Freeze-Drying of Proteins from a Sucrose-Glycine Excipient System: Effect of Formulation Composition on Initial Recovery of Protein Activity
February 2005
Wei Liu, D.Q. Wang, and Steven L. Nail
AAPS PharmSciTech
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Abstract
The purpose of this study was to investigate the impact 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 (LDH) and glucose 6-phosphate dehydrogenase (G6PDH) in a sucrose/glycine-based excipient system where the formulation composition was systematically varied. In a sucrose-only excipient system, activity recovery of both model proteins is about 80 percent, and is independent of sucrose concentration over a range from 1 to 40 mg/ml. However, when both sucrose and glycine are used and the ratio of the two excipients is varied, activity recovery decreases in a pattern that is consistent with inhibition of activity recovery by glycine crystals, despite the presence of adequate 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 glycine/sucrose 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.