Effects of Formulation and Process Variables on the Aggregation of Freeze-Dried Interleukin-6 (IL-6) After Lyophilization and on Storage

Abstract

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.

Effect of Collapse on the Stability of Freeze-Dried Recombinant Factor VIII and α-amylase

“…At the early time points the moisture content during storage was observed to be related to the storage temperature, with warmer storage temperatures producing higher cake moisture contents, however, at the later time points the moisture values at all storage temperatures reached similar levels. Transfer of water vapor from the rubber stopper to the lyophilized cake is known to occur, 2 and the pattern of increase in residual moisture observed here is consistent with this mechanism.…”

“…Although some investigations [2][3][4][5][6] have been published on the effects of collapse on freeze-dried materials, little has been published on the effects of collapse on the long-term storage stability of freeze-dried proteins. This study presents the results of an investigation of the long-term storage stability of two biologically active proteins formulated in the same excipient system and freezedried using three different freeze-drying protocols.…”

External Factors Affecting Protein Aggregation

“…169,372,373 Many formulation excipients have been used and have been found to be very effective in inhibiting protein aggregation during freeze -drying, especially sugars. These excipients include sucrose for rhIL -1ra, 237 both trehalose and sucrose for IL -6 374 and human factor XIII, 375 mannitol for a botulinum neurotoxin, 159 Tween 80 for bovine IgG 368 and rhIL -1ra, 237 Tween 20 and hydroxyethyl starch for a botulinum neurotoxin, 159 hydroxyethyl cellulose for aFGF, 376 and PEG for human FXIII. 375 Stabilization of protein by polymers is likely due to their capability of forming a glass with a higher transition temperature.…”

Stabilizing Effect of Four Types of Disaccharide on the Enzymatic Activity of Freeze-dried Lactate Dehydrogenase: Step by Step Evaluation from Freezing to Storage

“…Therefore, it is necessary to evaluate not only process stability but also storage stability of freeze-dried proteins. Previous studies have extensively investigated the effects of the composition of freeze-dried formulations [for example, their disaccharide (13)(14)(15)18,20,23,25,27,29,31) and moisture contents (23,26,28,32), and combination with other agents (12,15,16,18,21,22,25,29,30)] on the stabilization of proteins immediately after freezing or freeze-drying. The storage stability of the freeze-dried protein-disaccharide formulations has also been investigated under limited storage conditions (9,18,20,21,(30)(31)(32).…”