Peer-Reviewed Technical Note: Influence of Common Excipients on the Crystalline Modification of Freeze-Dried Mannitol

March 2, 2008
Pharmaceutical Technology, Pharmaceutical Technology-03-02-2008, Volume 32, Issue 3

Mannitol is the most commonly used bulking agent in freeze-drying formulation design. The benefit of using mannitol is that it crystallizes during freezing and permits drying processes at higher product temperatures, and thus with higher sublimation rates relative to purely amorphous systems (1). Mannitol, however, is known to form different crystalline modifications which compromises reproducibility of product characteristics and storage stability due to phase transformations (2, 3).


Mannitol is popular as a crystalline bulking agent in freeze-drying, but tends to form different crystalline modifications which may negatively impact storage stability. Mannitol-based pharmaceutical formulations usually contain additional formulation additives which may alter the extent of crystallization and the modification of mannitol. In this study, the impact of pure sucrose, trehalose, polysorbate 80, and citric acid as well as multi-component systems of these additives, on mannitol crystallization was investigated. Pure mannitol and 10 formulations with excipients were lyophilized using identical freeze-drying conditions. The product cakes from center and edge vials were characterized using Karl Fischer residual-moisture analysis, X-ray powder diffraction, and differential scanning calorimetry. The study revealed substantial differences in residual moisture content and significant changes in crystallinity induced by even small amounts of additives, especially in the case of sucrose.

Mannitol is the most commonly used bulking agent in freeze-drying formulation design. The benefit of using mannitol is that it crystallizes during freezing and permits drying processes at higher product temperatures, and thus with higher sublimation rates relative to purely amorphous systems (1). Mannitol, however, is known to form different crystalline modifications which compromises reproducibility of product characteristics and storage stability due to phase transformations (2, 3). The different modifications have been described extensively in the literature (4, 5). Because freeze-dried mannitol-based pharmaceuticals typically contain other excipients to fulfill the stabilization needs of the active pharmaceutical ingredient, the extent of crystallization and modification may be different than for pure mannitol (6).

This study investigates the influence of frequently used additives on the crystallization of mannitol. The additives used were: sucrose and trehalose, both widely used as lyoprotectants to stabilize proteins or peptides during drying; polysorbate 80, a surfactant frequently added to avoid surface denaturation of proteins during freezing; and citric acid, a popular buffer system (1).

Materials and methods

All chemicals used were analytical grade and obtained from Sigma Chemical Company (Munich, Germany). Pure mannitol and 10 mixtures with excipients were lyophilized during a single run (see Table I). Nine 10-mL vials from Lutz GmbH (Wertheim,Germany) for each formulation were filled with 3-mL solution. Total solid content of each mixture was kept constant at 50 mg/mL. Freeze-drying was performed using a laboratory scale Lyostar II freeze-dryer (FTS Systems, Stone Ridge, NY). The lyophilization cycle consisted of freezing at –40 °C, annealing at –15 °C for 3 h hours, and primary drying at –10° C and 100 mTorr (see Figure 1). Secondary drying was performed at 40 °C for 4 h. After the freeze-drying cycle, edge and center vials of each formulation were individually characterized using X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) (7). The residual moisture (RM) for one vial of each formulation was determined by Karl Fischer analysis.

Figure 1

Residual moisture measurements. Residual moisture was determined by coulometric Karl-Fischer titration using a water vaporizer (Mitsubishi Chemical Company CA-06 with VA-06, Tokyo, Japan). Approximately 35–90 mg of the solid samples were transferred to the vaporizer unit where the sample was heated to 140° C. Extra dry nitrogen was used as a carrier to transfer the moisture into the measurement cell.

X-ray powder diffraction. A 30–50 mg sample was weighed into the XRPD sample holder, compacted in the sample block, and examined using an X'Pert MPD(Royal Philips, The Netherlands)with Cu K α radiation at 40kV/40mA and 25 °C. Scans were measured in the range 2θ = 0.5°–40° C with a step size of 0.02 °C. The diffractograms were plotted in Microcal Origin 7.5 (Originlab, Massachussettes), and the baseline corrected for a measurement offset.

Table I: Binary and ternary mixtures used.

Differential scanning calorimetry. The authors used a Mettler Toledo 822 DSC with STARe SW 9.01 software (Mettler Toledo, Switzerland) for thermal analysis. Approximately 20 mg of sample was weighed into a 40 μL aluminum pan and compressed in a dry-nitrogen atmosphere. Pans were then sealed hermetically and transferred into the DSC cell. Two different methods were applied. First, all formulations were scanned with a ramp rate of 10 °C/min from 0 °C–180 °C to reveal potential glass transitions, melting events, or recrystallization. A lower ramp rate (2.5 °C/min) was used for some formulations showing inconclusive results at fast ramp rates.

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 mannitol cake.

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).

Figure 2

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.

Figure 3

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.

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,

*To whom all correspondence should be addressed.

Submitted: Jan. 23, 2008. Accepted: Feb. 1, 2008.


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