Compared with biopharmaceuticals, most freeze-dried small-molecule drugs are relatively inexpensive. To make their manufacture
profitable, a high throughput without compromising the final quality is indispensable. The best approach to an optimised freeze-drying
cycle is the reduction of primary drying time because this phase is commonly the most time-consuming part (1, 2). To follow
this approach, an accurate and representative measurement of the critical formulation temperature (CFT) is required, which
poses the upper boundary for the product temperature at the sublimation interface (Tp) during primary drying (2,3). With regard to process time and product-quality attributes, such as elegantcake appearance,
low residual moisture, quick and complete reconstitution and drug activity, the product temperature over time profile during
primary drying should be close to but below the CFT (2–4). In some cases, however, drying in the microcollapse regime is possible
without severe structural loss, as reported for protein formulations, and could also be a promising approach to further process
optimisation for small molecule drugs (5–7). In this study, the thermal properties of gentamicin sulphate as a small-molecule
model substance were characterised by means of freeze-dry microscopy (FDM) and differential scanning calorimetry (DSC) to
determine the CFT at different concentrations and to investigate the potential for further freeze-drying cycle optimisation.
Materials and methods
The pure gentamicin sulphate drug substance used for the thermal characterisation was provided by Merck KGaA. Gentamicin sulphate
solutions of 2, 5, 10, 20 and 30% (w/v) were prepared with deionised water.
DSC. The glass-transition temperature of the maximally freezeconcentrated solute (Tg') for the 5% (w/v) gentamicin sulphate solution was determined using a differential scanning calorimeter (DSC822e, Mettler Toledo). Data analysis was conducted with software (STARe Software V 9.01, Mettler Toledo) and values were
provided as "onset" and "midpoint" (i.e., half height) of the transition. 30 µL of sample solution were hermetically sealed
in a 40-µL aluminum pan, cooled down to -80 °C at a cooling rate of 5 °C/min, equilibrated for 10 min and reheated at 10,
3, and 1 °C/min. For one sample measured with a heating rate of 3 °C/min, an annealing step was implemented for 90 min at
-20°C. Nitrogen was used to purge the measuring cell throughout the experiment.
FDM. Collapse temperatures (Tc) for the 2, 5, 10, 20 and 30% (w/v) sample solutions were investigated directly after preparation and after one week of storage
at room temperature. Validation of the temperature sensor and classification of collapse behaviour were conducted according
to the literature and results are reported as an average of four measurements (8). The FDM equipment consisted of a microscope
(Zeiss Imager.A1 microscope, Zeiss) with a polariser, a lambda disk and a freeze-drying stage (FDCS 196, Linkam Scientific
Instruments). For each measurement, a sample volume of 2 µL was pipetted onto a cover glass lying on the silver block oven
of the stage. A smaller cover glass was placed on top of this drop. Constant layer thickness was assured by using custom-made
metal pieces (i.e., 25 µm) as spacers. To improve thermal contact between the bottom cover glass and the oven, a droplet of
silicon oil was added. The sample droplet was frozen at a rate of 1.0 °C/min and equilibrated at -45°C for 10–13 min depending
on the velocity of the sublimation interface. After 8 min of equilibration, the vacuum pump was switched on and sublimation
could subsequently be observed. The sample was heated at a rate of 1.0 °C/min following the equilibration step. The magnification
used during the experiments was 200-fold. Pictures were captured by the software in 1-second intervals using a digital camera
(Pixelink, 1.3 MP) and analysed with the LinkSys 32 software (Linkam Scientific Instruments). The stage was purged with dry
nitrogen during freezing and heating. Pressure was measured using a calibrated Pirani gauge (Linkam Scientific Instruments)
The maximum achievable vacuum (< 0.03 mbar) was kept constant throughout the measurement.