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Why and how to carry out thermal characterization of a product before lyophilization?

In this interesting article, Antoine Babin of Biopharma Technologies France provives an overview about why thermal characterization in your freeze-dried products is so important.


During the stages that precede lyophilization of a product, it is important to characterize it as fully as possible. Depending on its nature, a whole battery of analyses will be conducted which will identify its size, its shape, its concentration, its structure, its activity; or any other information which will allow it to be understood in the smallest detail.


The golden rule in lyophilization is never to add an ingredient if its value in the lyophilization cycle has not been clearly determined. Thermal characterization contributes to the understanding of the behavior of the product during the lyophilization cycle. Why carry out thermal characterization of the product? Lyophilization consists of a water sublimation process. By definition, the water must be solid, therefore frozen before being sublimated. But what about the solute! Current standards impose cosmetic criteria on the lyophilized cake. Broadly, the lyophilized product must resemble a very compact powder, if possible adherent to the white walls, etc.

In lyophilization, this collapse generally happens when the product exceeds a certain temperature during the primary drying phase, that is the phase in which the solution water will be removed. If I do not know this temperature, which is specific to the product, then it will be difficult to determine the ideal shelf temperature and the working pressure that must be used during the lyophilization cycle. Indeed, it is commonly accepted that the ideal working pressure in the lyophilizer chamber is between 20 and 50% of the pressure of steam at the surface of the lyophilization cake. This steam pressure is dependent on temperature; without knowing this, it is difficult to determine the ideal pressure.

We note also that a correctly composed lyophilization cake that is not collapsed will also have an impact on the efficacy of secondary drying and the said extraction of water associated with the product. It is also possible to note, in the case of collapse, a slight coloration of the lyophilizate caused by Maillard reactions, let us say a slight caramelization of the product, which is obviously not desired.

Which temperatures should be characterized? Ideally we are seeking to know from which temperature in the product we risk observing the collapse of the lyophilization cake. In other words, when and why the cheese soufflé will deflate when it comes out of the oven! 3 temperatures must be defined: the eutectic temperature ET, the glass transition temperature Tg’ and the collapse temperature TC.


The ET defines exclusively a crystalline product and the Tg’ characterizes an amorphous product. There is no need to know precisely the difference between crystalline and amorphous. However and to use another image, if I have a pallet of well stacked bricks delivered to my house, the assembly is crystalline; I push the bricks into my garden, the assembly is amorphous even if individually each brick remains crystalline.


By experience, less than one product in 100 that we analyze in our laboratory displays a crystalline characteristic therefore an ET! We note in passing that often, the term “eutectic temperature” is used to speak of the critical temperature; which in most cases represents an error of language since we should rather speak by default of Tg’ therefore of the glass transition temperature!


Should we speak of eutectic melting or viscous flow? If the product is crystalline, and consequently an ET has been defined, then, if the temperature at the center of the product exceeds the ET during primary drying, we will speak of eutectic melting. This is the example of our scaffolding which will collapse suddenly. The product will doubtless resemble a caramel at the bottom of a well, probably with droplets at the edge of containers above the initial filling level, a sign of partial boiling during the cycle.

If the product is amorphous, the most probable case, then a Tg’ has been defined and we will speak of viscous flow. The product will soften with increasing temperature, rather like a piece of modeling clay that is being stretched would do; it will progressively extend until it’s possibly breaks. The product then displays non-regular reductions in diameter across the whole of the cake for example.

When we measure a Tg’, depending on the method and equipment, there may be slight variability. Either I take into account the moment when I feel that the resistance of my modeling clay is beginning to give way (onset), or the moment when I no longer feel the resistance of the clay but it continues to stretch without breaking (offset). Most often, the analysis suggests to us a midpoint Tg’ measurement, that is between onset and offset. Whatever the case, it is important to note with regard to an amorphous product, that if there is a collapse, then it will necessarily occur after the analyzed Tg’ value. Therefore, in order to be able to define the highest possible collapse temperature (to be able to work at the highest possible shelf temperature), it is preferable to accompany the Tg’ analysis with an additional analysis.

It involves determining the collapse temperature Tc which will be necessarily equivalent to or higher than the Tg’, in order not to deprive oneself of the possibility of gaining several °C during the cycle, without however affecting the quality of the lyophilization cake.

What are the different methods of thermal characterization? The most common method used to determine the nature of the product (ET or Tg’), is probably DSC (Differential Scanning Calorimetry). DSC measures the flow of heat “in & out” of a sample over a temperature range in comparison with a reference sample, most often empty.


Currently, modulated DSC “mDSC” will generally be preferred to DSC. mDSC allows the generation of temperature ramps with a regular wave that enables the differentiation of reversible thermal transitions (glass transition) from non-reversible transitions (eutectic melting). MDSC will therefore allow the dissociation of thermal events within the product which will not be observable in classic DSC. mDSC effectively allows the separation of concurrent events; observable for example with histidine, mannitol or glycine.


Complete article in "La Vague" April 2022 - Why and how to carry out thermal characterization of a product before lyophilization?