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
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.
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.