The preservation of lyophilized Human Growth Hormone activity: how do buffers and sugars interact?

PURPOSE: One of the most common classes of excipients used in protein formulations are buffers. The aim of this work is to investigate the effect of buffers on protein stabilization given by sugars during freeze drying. METHODS: Molecular Dynamics simulations of human growth hormone (hGH) in the presence of sucrose and trehalose were performed, and the impact of phosphate and citrate buffers on their protective action was analyzed. RESULTS: We found that buffers broke the hydrogen bonding network formed by excipients, and the consequences of this disruption of structure ordering were different in sucrose-based or trehalose-based formulations. More specifically, we observed that buffers increased protein recovery in the presence of excipients, such as sucrose, that exert their action mainly by preferential exclusion. By contrast, the opposite effect was sometimes noted in the case of excipients, such as trehalose, whose protective action is related to the formation of a highly structured matrix. CONCLUSIONS: We found that buffers have important properties, other than the control of pH, that can contribute to the overall stability of proteins. Some of these properties are related to their interaction with the other components of the formulation.