The mDSC analysis of the mannitol solution (1) shows a single crystallization event on the non reversing heat flow line at -26.5°C with a peak at -23.6°C then melting of the solvent at -2.6°C (Figure 1). While its DSC450 analysis shows a crystallization event at -25.3°C with a peak at -24.2°C then melting of the solvent at -3°C (Figure 2). Which is completely comparable.

The mDSC analysis (2) of the mannitol/glycine solution revealed several events starting with the onset of the glycine glass transition at -46.3°C; and therefore, a change in mobility which caused crystallization of the glycine at -43.8°C. The glass transition of mannitol then began at around -34.8°C then its crystallization at -34.1°C. The DSC450 revealed only the two crystallization events at -43.7°C and -33.5°C respectively, events almost concurrent with the two glass transitions, making them very difficult to observe.

There is a third possibility, DTA-impedance measurement (Differential Thermal Analysis and impedance analysis). This analysis is conducted on a Lyotherm, a device developed by Biopharma Process Systems and the  late Professor Louis Rey, that in the 2000s, transferred its lyophilization activity to the Centre de Ressources Technologiques, Aerial.

DTA operates on the same principle as DSC but measures the temperature differential in place of heat flow. The Lyotherm also measures the impedance value (Z) which among other things, allows the observation of other transitions of the product potentially the cause of micro events such as micro collapses for example. This is why we prefer this method since we will draw more information from it.

In this example we observe on the pink impedance line the beginning of the softening of the frozen matter that the red DTA line would not have let us anticipate.  It happens in fact that the increased mobility of the material detected in impedance analysis appears at a temperature significantly lower than the detection of the collapse temperature Tc measured by cryomicroscopy as shown in the table below.

1 to 2μL of sample is deposited on the quartz slide mounted on a silver block. The sample is then covered with a glass coverslip above a 70μm spacer as follows:

The sample is then stabilized at low temperature, generally -50°C, then a vacuum is created in the cell to generate the dry matter on the sublimation front. The temperature is then gradually increased until a deterioration of the sublimation front is induced. This is detected visually, but there are now image analysis software options that automatically determine the temperature from which deterioration of the sublimation front appears.

It is possible, from the observation made in Figure 5, to lower the temperature of the plate again to re-establish the structure of the frozen sample to refine the reading of the collapse temperature and so to define it with greater accuracy as presented in Figure 6.

Cryomicroscopy dedicated to lyophilization has other benefits. For example, it will allow visualization of micro collapses in mixtures. This can occur particularly during eutectic melting of crystalline elements within a rigid amorphous structure, or the reverse, that is to say viscous flow of amorphous elements within a rigid crystalline structure.

Figure 7 shows a glucose 1%/mannitol 2% mixture where we can see areas of micro collapse at around -41°C, that is the temperature close to the approximate Tc of glucose.

Crust formations can be also observed on the product surface. As in the example in Figure 7b.

Some materials may display several critical temperatures and coexist naturally in amorphous and crystalline form. They may be defined in the literature as being metastable. Mannitol displays this characteristic and while we estimate its glass transition temperature Tg’ to be in the vicinity of -32°C, as it is very difficult to measure, we know the eutectic temperature of its crystalline form at -1.4°C. We therefore understand the benefit of working in lyophilization with crystalline mannitol which will allow us to envisage higher working temperatures and therefore potentially a shorter cycle duration. In this type of situation it may be of value to envisage an “annealing” consisting in:

1) reducing the temperature below the Tg’,
2) stabilizing,
3) raising the temperature to a point between the Tg’ and the ET,
4) stabilizing.

The annealing carried out in this way should make it possible to opt for thermal processing of mannitol in its crystalline form.

If your cryomicroscope is equipped with a polarizer then the formation of the crystalline form will generate an observable color change. It is therefore possible to use the cryomicroscope to establish the potential benefit (or not) of carrying out annealing, at different temperatures.

The example in Figure 8 is a mannitol solution that was first frozen then brought to -5°C.