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Table I: Tg’ values (onset and midpoint) from differential scanning calorimetry measurements for 5% (w/v) gentamicin sulfate
solution determined with different heating rates.
DSC. As reported in the literature, Tg´ does not significantly change with concentration because the composition of the maximally freeze-concentrated solute is
independent of the initial concentration (9). For this reason, only the 5% (w/v) solution was investigated by DSC. At first,
the sample solution was scanned with a 10 °C/min heating rate to roughly detect the temperature range of Tg´. A fast heating rate increases sensitivity but can also impair representativeness of the measurement by shifting Tg´ to higher temperatures (9). To obtain the true result, additional heating rates of 3 and 1 °C/min were applied. Results
are summarized in Table I, where the Tg´ onset and midpoint values are listed to illustrate the magnitude of the transition. The implementation of an annealing step
revealed a glass transition that was comparable to the experiment without annealing regarding extend and temperature range.
Crystallization of gentamicin sulfate, therefore is not expected.
Figure 1: Freeze-dry microscopy pictures of the collapse behavior of 5% (w/v) gentamicin sulfate solution: (a): intact dried
matrix, no collapse; (b) onset of collapse, arrows indicate first visible changes; and (c) full collapse, loss of coherent
product matrix.
FDM. The collapse behavior of gentamicin sulfate solution is exemplarily shown in Figure 1 for the 5% (w/v) sample. Figure 1(a)
shows the typical appearance of the drying sample at a temperature where no collapse occurred yet. The dark and dense matrix
on the left represents the already dried amorphous matrix while the colorful area on the right shows the frozen structure.
With proceeding drying, the sublimation interface progresses, in this case, from left to right. The temperature of the onset
of collapse (Toc) is reached when the first structural changes can be visibly detected by formation of little pink holes or fissures in the
dried structure adjacent to the sublimation interface as a result of viscous flow (see Figure 1(b)). During ongoing sublimation,
the initial holes and gaps grow until the temperature of full collapse (Tfc) is reached, where the structural loss is so severe that there is no coherent dried-product matrix any more (see Figure 1(c)).
Table II summarizes the results of the FDM measurements. As reported earlier, Tc is not a material constant but depends on concentration (3, 10, 11). In the case of gentamicin sulfate, an increase of approximately
3 °C can be observed in the range of 2 to 30% total solid content. The gap between Toc and Tfc (also denoted as the microcollapse regime) can offer valuable information about the temperature tolerance of the product
(5). Depending on the formulation, this temperature range can widen up to 10 or 15 °C (7, 8, 12), and recent publications
describe primary drying in the microcollapse region without significant macroscopic structural loss (5–7). With a difference
of about 2 °C, the gap between Toc and Tfc for the presented measurements is relatively small and does not increase distinctly with concentration.
Table II: Average values (n = 4) of collapse temperatures for gentamicin sulfate solutions of different total solid content,
obtained by freeze-dry microscopy measurements directly after preparation and after one week of storage at room temperature.
Toc is temperature of the onset of collapse; Tfc is temperature of full collapse; Tc-50 is calculated "midpoint" collapse (numerical average of Toc and Tfc). Sdv is standard deviation.
FDM measurements were repeated after one week of storage at room temperature to investigate potential instability of the solution
that may lead to changes in Tc. Regarding the standard deviations, no clear difference between immediate measurements and experiments after one week of
storage can be observed. It is important to note that at higher concentrations, the visual detection of collapse becomes more
difficult. Due to the increased density of the sample and, therefore, reduced translucence, variations between replicate measurements
increase.
Figure 2: Comparison of critical formulation temperatures of gentamicin sulfate solutions with different total solid content.
Half-filled symbols represent Tg´ (differential scanning calorimetry. Open symbols represent Tc (freeze-dry microscopy) directly after preparation. Filled symbols represent Tc (freeze-dry microscopy) after one week of storage at room temperature. Squares represent onset of collapse or glass transition.
Circles represent midpoint of collapse or glass transition, and triangles represent full collapse or endset of glass transition.
Comparison of DSC and FDM data. Tg´ and Tc are not identical as the measurement methodologies are different. Collapse describes a dynamic process because sublimation
actually takes place during a FDM experiment, and the observed structural changes occur in the already dried matrix. In contrast,
during the DSC measurement, the amorphous drug is in direct contact with ice without a drying process. Therefore, the experimental
conditions of a FDM measurement better simulate those of a real freeze-drying run, and the structural changes can be visually
observed. Moreover, Tg´ values are commonly reported as midpoint while the collapse temperature mostly refers to the onset of collapse. To roughly
estimate a 50% structural loss in the dried matrix (midpoint) and to simplify comparison to DSC data, Tc-50 was introduced as a calculated average of Toc and Tfc (8). Figure 2 illustrates the CFTs determined with both methods during the present study. As previously described in the
literature (2, 8, 12), Tg´ in the authors' study is lower than Toc, with greater difference at higher total solid content. For example, Toc of the 5% solution is about 2 °C higher, and at 30% total solid content, the difference amounts to approximately 4 °C compared
to the midpoint Tg´. Taking Tc-50 into account, the gap ranges from 2.6 °C (5% w/v) to about 5 °C at 30% w/v for the measurement series directly after preparation.
Regarding the fact that an increase of only 1 °C in product temperature during primary drying can reduce cycle time up to
13%, the application of Toc instead of Tg´ clearly permits more space for cycle optimization, especially at higher concentrations (13).
Henning Gieseler, PhD, works in the Department of Pharmaceutics at the University of Erlangen, Erlangen, Germany 91058.
tel. +49 9131 85 29545, gieseler@freeze-drying.eu.
Articles by Henning Gieseler, PhD
