DETERMINING THE WATER SORPTION MONOLAYER OF LYOPHILIZED PHARMACEUTICAL PROTEINS

The concept of monolayer water coverage is useful in the development o f lyophilized protein formulations. Herein, we have explored three dif ferent methodologies to determine the water monolayer for pharmaceutic al proteins: (1) theoretical prediction based on the amino acid compos ition and their relative propensities to sorb water; (2) a traditional adsorption isotherm measurement by Karl Fischer water titration of sa mples held at various relative humidities (created by saturated salt s olutions); and (3) an adsorption isotherm measurement with a gravimetr ic sorption analyzer (GSA), which consists of a microbalance within a computer-controlled humidified environment. Data from the latter two m ethods were analyzed with the Brunauer-Emmett-Teller (BET) gas adsorpt ion equation to yield experimental monolayers. In our study, we examin ed 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 monolaye r for a model sugar (trehalose) and polyol (mannitol), which are poten tial 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 up on lyophilization. Mannitol tended to form anhydrous crystals upon fre eze-drying, and was thus not amenable to analysis. The utility of both traditional and GSA methods for determining the water monolayer was e xtended to colyophilized protein:sugar systems as well.