Determining the water sorption monolayer of lyophilized pharmaceutical proteins.

The concept of monolayer water coverage is useful in the development of lyophilized protein formulations. Herein, we have explored three different methodologies to determine the water monolayer for pharmaceutical proteins: (1) theoretical prediction based on the amino acid composition and their relative propensities to sorb water; (2) a traditional adsorption isotherm measurement by Karl Fischer water titration of samples held at various relative humidities (created by saturated salt solutions); and (3) an adsorption isotherm measurement with a gravimetric sorption analyzer (GSA), which consists of a microbalance within a computer-controlled humidified environment. Data from the latter two methods were analyzed with the Brunauer-Emmett-Teller (BET) gas adsorption equation to yield experimental monolayers. In our study, we examined six different therapeutic proteins and found that for each case all three approaches yielded similar results for the water monolayer. We also attempted to use the BET equation to determine the water monolayer for a model sugar (trehalose) and polyol (mannitol), which are potential excipients in pharmaceutical protein formulations. We found that calculations from the data obtained by the traditional and GSA methods yielded consistent results for trehalose, which remained amorphous upon lyophilization. Mannitol tended to form anhydrous crystals upon freezedrying, and was thus not amenable to analysis. The utility of both traditional and GSA methods for determining the water monolayer was extended to colyophilized protein:sugar systems as well.