Results and discussion
Residual moisture variability. As expected, the pure mannitol cake contained low levels of residual moisture, approximately 0.2% (w/w), due to its fullly
crystalline character. Surprisingly, the binary mixtures of mannitol and polysorbate 80 showed even lower levels of moisture
than Formulation 1; citric acid showed comparable moisture levels. Low levels of sucrose, however, led to substantially elevated
RM results (e.g. 1.0% for Formulation 2 and 2.4% for Formulation 3). This trend can also be observed in the ternary mixtures
9 (1.7%) and 10 (2.0%). The amorphous phase consisting of a share of mannitol as well as sucrose retains higher concentrations
of residual water. Here, the increased RM may be due to microcollapse of the amorphous phase onto the crystalline mannitol
during primary drying, which restricts additional removal of water (8). The RM elevation can also be observed for trehalose,
but to a lesser extent: both Formulation 4 and Formulation 11 contained 0.3% RM which is only slightly more than the pure
Table II: Summary of analytical results.
XRPD. The crystalline peaks in the diffractograms were compared to reference diffractograms of pure α-, β- and δ-mannitol and of
mannitol hydrate reported in the literature (9-11). Note that the modification ratio was determined semi-quantitatively. Center
and edge vials of each formulation showed the same qualitative composition for all formulations, excluding radiation effects
and drying variations as possible factors for modification differences. The mannitol used for the solutions consisted of pure
δ-modification. In contrast, practically all of the lyophilized formulations showed one main modification and some amount
of one or more other modifications. Formulation 1 consisted of mostly β-mannitol and showed trace amounts of α- and δ-mannitol
(see Figure 2). Similar compositions with higher amounts of α-modification were observed in formulations containing mannitol
and citric acid (see Table I: Formulations 7 and 8).
The addition of sucrose or trehalose led to an increased amorphous fraction of the cake and also affected the crystalline
modifications. Mixtures with 0.5% and 1% sucrose showed mostly δ-mannitol and some α- and β-mannitol, but also contained significant
amounts of mannitol hydrate, which corresponded well with the increased moisture content (see Figure 3). In Formulation 4
and Formulations 9–11, the δ- modification was dominant; the formulations containing sucrose also showed peaks of mannitol
hydrate. Formulation 11 also contained some mannitol hydrate, which indicates effects of the increased amorphous fraction.
Note that it was not possible to detect crystalline citric acid which might have remained amorphous during freeze-drying.
Thermal analysis. The thermal analysis did not allow quantitative determination of mannitol modifications, but it was possible to obtain valuable
information. In the majority of the formulations, the amorphous phase consisted mainly of sucrose, trehalose, or polysorbate
80and showed only one glass transition (Tg). The crystallization peaks gave an indication of the amount of mannitol that remained
amorphous during lyophilization. Formulations with high moisture content displayed an additional small endothermic event at
130° C followed by an exotherm which could be caused by conversion of mannitol hydrate to an anhydrous modification. The melting
point of pure α- and β-mannitol have been described as 166 °C and 166.5 °C, which clearly makes a separation impossible(12).
Reports for the melting point of δ-mannitol vary from 150–158 °C, which is sufficiently lower than the others to allow identification
(5). In the thermograms of the formulations studied, there were often smaller melting peaks of δ-mannitol followed by recrystallization
and the melting peak of α- and β-mannitol which could be isolated using the slower heating rate. A sample thermogram is shown
in Figure 4.
The authors were able to lyophilize solutions of pure mannitol as well as various combinations of mannitol with excipients.
The product cakes were characterized regarding residual moisture, crystallinity, and thermal characteristics. The excipients
caused significant differences in physical properties, especially in the case of sucrose. Further investigation is required
to identify patterns and consequences of these influences, in particular with an active pharmaceutical ingredient (protein
or peptide). This study, however, clearly indicates that the effects of additives on crystallization of mannitol must be taken
into account during formulation development of freeze-dried products.
Stefan Schneid, Xenia Riegger, and Henning Gieseler, PhD* work in the Department of Pharmaceutics at the University of Erlangen, Erlangen, Germany 91058. tel. +49 9131 85 29545, firstname.lastname@example.org
*To whom all correspondence should be addressed.
Submitted: Jan. 23, 2008. Accepted: Feb. 1, 2008.