Formulations of sugars with amino acids or mannitol--influence of concentration ratio on the properties of the freeze-concentrate and the lyophilizate.

Formulations consisting of either sucrose or trehalose with glycine, lysine-HCl, or mannitol were studied to determine how the ratio of the excipients affects the design of the lyophilization program and the properties of the final cake. Glass transitions (Tg', Tg), crystallization temperatures, and eutectic melting temperatures were measured by differential scanning calorimetry, the physical state of the excipients was determined by x-ray powder diffraction, and residual moisture was measured by Karl Fischer titration. The addition of increasing amounts of glycine, lysine-HCl, or mannitol to a sucrose solution caused a progressive depression of the Tg', an effect that was more pronounced with the amino acids. In equivalent ratios with sucrose, the two amino acids induced a comparable Tg' shift due to their low Tg' values. For lysine-HCl, two apparent Tg' with midpoint temperatures of -69 and -56 degrees C were measured. For mannitol, the Tg' depression was unexpected because mannitol exhibits a higher Tg' than that of sucrose. During lyophilization, the ratio of the amorphous amino acids or mannitol to the sugar determined whether crystallization could be induced by an annealing step performed after freezing. Crystallization could be verified by a shift of the formerly depressed Tg' back to the value of the sugar and by the detection of the eutectic melting peak of the crystallized compound. The crystallized excipients served as excellent bulking agents. In the freeze-dried cake, amorphous glycine and even more amorphous mannitol lowered the Tg value. If the cake was stored above Tg, subsequent crystallization of mannitol occurred. The results emphasize that the qualitative and quantitative composition of a formulation has profound implications on the design of a lyophilization program and on the characteristics of the freeze-dried cake.

Effects of formulation and process variables on the aggregation of freeze-dried interleukin-6 (IL-6) after lyophilization and on storage.

This study assessed the impact of residual moisture, Tg, and excipient physical state of different formulations on the "in-process" and shelf-life stability of freeze-dried interleukin-6 (IL-6). The effect of an annealing procedure was also evaluated. Characterization of the lyophilizates was done by Karl Fischer titration, differential scanning calorimetry (DSC), and x-ray measurements. Analysis of protein stability was carried out by size exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and turbidity measurements. During freeze-drying, the most effective protection against aggregation was provided by completely amorphous formulations consisting of trehalose or sucrose either alone or in combination with glycine or mannitol. Other amorphous formulations like those of sucrose with lysine-HCl or dextran could not provide comparable stabilization. In lyophilizates containing a crystallized excipient such as glycine or mannitol, IL-6 suffered destabilization, which was less pronounced if an additional amorphous excipient was present. For the completely amorphous formulations, aggregation was prevented during a 9-month storage at 25 and 40 degrees C as long as the storage temperature did not exceed the Tg value of the lyophilizate, otherwise severe damage occurred. Formulations containing amorphous dextran or lysine-HCl could not effectively stabilize IL-6 even when stored below Tg. Annealing helped to improve cake robustness and appearance, but for lyophilizates containing an excipient crystallized by annealing an increase of IL-6 aggregation was observed despite a storage below Tg. Thus, the amorphous state of the excipients and a high Tg can be considered necessary conditions for preventing aggregation of freeze-dried IL-6. Whether the conditions are also sufficient depends on the choice of excipients. Destabilization can occur with some excipients despite their amorphous state as well as in the presence of crystallized excipients despite a storage below Tg. Compared to sucrose, trehalose is a more favorable excipient for protein lyophilization because it exhibits a higher Tg, possesses better stabilizing properties, and can reduce protein aggregation which may have been caused by annealing.