The impact of protein concentration on mannitol and sodium chloride crystallinity and polymorphism upon lyophilization.

The effect of protein concentration, in the range of 0-26 mg/mL for two Fc-fusion proteins, on the crystallinity and polymorphism of mannitol and sodium chloride in a lyophilized model formulation was examined. Mannitol hydrate levels were quantified based on moisture data and correlated to the X-ray diffraction peak area. In all formulation conditions, sodium chloride did not crystallize in samples with >44% total amorphous content. As protein concentration increased through the range of 1-5 mg/mL prior to lyophilization, beta-mannitol decreased in amount, becoming undetectable at protein concentrations above 5 mg/mL. Conversely, delta-mannitol increased as a function of protein concentration, reaching a maximum level at approximately 5 mg/mL protein. Above 10 mg/mL protein, mannitol crystallization was increasingly inhibited. Sucrose control vials showed higher levels of mannitol hydrate than either model protein. Both proteins behaved comparably with respect to mannitol crystallinity and polymorphism despite significant differences in molecular weight. Because of the differences between protein and sucrose control samples, protein concentration must be taken into consideration when assessing the lyophilization of mannitol containing solutions.