Temperature Control

As technology within the analysis sphere continues to evolve, temperature control is becoming increasingly important for drug discovery and research

Dr Duncan Stacey at Linkam Scientific Instruments

In many areas of the pharmaceutical industry, it is becoming increasingly important to understand the micro- and thermo-mechanical properties of a substance. Temperature-controlled microscopy covers a wide range of analytical techniques, including methods that involve combining traditional thermo-analytical methods, such as differential scanning calorimetry, and image analysis techniques, such as thermal analysis by structural characterisation (TASC), to characterise pharmaceuticals and their component materials (1, 2). Temperaturecontrolled microscopy takes two key forms: hot-stage microscopy (HSM) which allows researchers to understand the effects of higher temperatures on a sample; and cryo-microscopy, which allows researchers to analyse how substances respond to sub-zero temperatures.

In this article, we will take a look at the importance of temperature in understanding the behaviour and potential uses of different pharmaceutical and biopharmaceutical substances, and how temperature-controlled microscopy stages are enabling researchers to advance drug discovery and development studies.

In pharma and biopharma research, temperature control provides a wealth of useful information, such as obtaining data on the changes in morphology of pharmaceutically relevant compounds, including active pharmaceutical ingredients (APIs), and interactions with the excipients.

HSM allows researchers to observe transitions that take place within a sample, such as melting/boiling points, desolvation, polymorphisms, glass transitions, and the crystallisation process, as well as other phase changes (1). Conversely, cryo-temperature testing can also provide a plethora of information for pharmaceutical analysis.

Electron microscopy (EM) enables molecules to be studied in a variety of functional states, using tiny amounts of material (at almost atomic resolution). Cryo-EM uses electron beams in vacuum conditions at cryogenic temperatures to overcome the challenge of measuring biological specimens with high levels of water present.

Cryo-correlative light and electron microscopy (CLEM) goes a step further, and brings the advantages of cryo-EM together with low temperature fluorescence, to increase sensitivity for the detection of chemical, biological, and genetic processes inside live cells. Cryo-CLEM enables direct fluorescent labelling and targeting of molecules or molecular assemblies in cryo-immobilised samples, pinpointing regions for subsequent high-resolution imaging using EM.

Humidity is also a key parameter to be considered within temperature-controlled microscopy. Combining temperature-controlled microscopy with control of the humidity of the environment allows researchers to also bring in the effects of humidity on a substance, and understand how this may impact the behaviour and degradation of materials. Controlling heat and humidity allows researchers to create an environment within which to observe notable changes, and this level of analysis into how samples and materials behave under certain conditions offers a better understanding of how they perform in real-world environments. In pharmaceuticals, this insight allows researchers to understand details including how to maintain drug efficacy, such as ensuring finished products and APIs are stored in the right conditions, before going to market.

There are many examples where researchers have included temperature as a testing parameter in their pharmaceutical analysis. At the University of Glasgow, Scotland, researchers used HSM in the study of phase transitions and crystallisation processes of mixed liquid systems, as controlling crystallisation is especially important when developing medications such as therapeutic proteins. This is because small tweaks to solvents can lead to issues such as the formation of different polymorphs, or failure of the protein to crystallise altogether.

Results from experiments such as this are consequently of interest to biopharma developers. Using temperature control as a testing parameter allowed the team to observe small changes within a sample and predict how the substance would respond, meaning that developers may, in future, be able to retain some element of control over these factors (3).

Cryo-temperatures have played their role in pharmaceutical research as well. Using fluorescence microscopy, cryo-soft X-ray tomography (cryo-SXT), and transmission electron microscopy, a group of researchers and clinicians in Barcelona, Spain, observed the effectiveness of cisplatin, an anti-cancer drug, at extremely low concentrations, to establish the lowest possible dose needed to produce an effect and minimise toxicity (4). Using a fluorescence microscope, the group imaged cryogenically frozen cell samples, keeping them vitrified at liquid nitrogen temperatures. The samples were then analysed using cryo-SXT, which enabled 3D investigation on a nanometric scale. These methods gave promising results, showing that tricine – one of two adjuvants studied – facilitated effective therapeutic use of cisplatin at lower doses than previously applied, possibly paving the way for the advancement of chemotherapy treatments with diminished side effects for patients.

Another key example of temperature control in action in pharmaceutical analysis is a research group at the National Institute for Biological Standards and Control, UK, which is working to understand the complexities of freeze-drying (lyophilisation) pharmaceuticals using advanced freeze-drying microscopy (FDM) techniques. Lyophilisation is a useful technique in drug development, as it allows researchers to reduce the risk of degradation and improve stability of pharmaceutical products by removing the presence of water. However, lyophilisation as a process can be lengthy and complex, and FDM allows researchers to better understand this method, and establish how drug products will behave under different thermal conditions.

The group, led by Dr Paul Matejtschuk, is focusing on optimising the formulations of freeze-dried liposomal drugs, which pose developmental challenges due to their chemical and physical instability. By using a specialised cryo-stage mounted on an optical microscope, Dr Matejtschuk and his team can predict the ideal freeze-drying conditions for liposome-cryoprotectant mixtures, by assessing the freezing, collapse, and melt temperatures (5). Experiments such as this are vital in the continued effort to develop fast, transferable, and scalable freeze-drying methods to stabilise pharmaceutical compounds.

The techniques described in this article highlight a selection of the many high-heat and cryo-methods available that are helping to advance pharmaceutical research. Using dedicated temperature-controlled stages that are compatible with a wide range of microscopy techniques, such as FDM and TASC, pharma researchers are provided with the necessary tools for a vast number of pharmaceutical applications, from evaluating new therapies to understanding new vehicles for drug delivery, and beyond.

References

1. Kumar A, Singh P, Nanda A, Hot stage microscopy and its applications in pharmaceutical characterization. Appl Microsc, 50(1):12. doi:10.1186/s42649-020- 00032-9, 2020.

2. Reading M, Qi S, Alhijjaj M, Local Thermal Analysis by Structural Characterization (TASC). In: Šesták J, Hubík P, Mareš J (eds) Thermal Physics and Thermal Analysis. Hot Topics in Thermal Analysis and Calorimetry, 11. Springer, Cham, 2017.

3. Walton F and Wynne K, Control over phase separation and nucleation using a laser-tweezing potential. Nature Chem 10, pp506–510, 2018.

4. Gil S, Solano E, Martinez-Trucharte F, et al, Multiparametric analysis of the effectiveness of cisplatin on cutaneous squamous carcinoma cells using two different types of adjuvants. PLoS ONE, 15(3): e0230022, 2020.

5. Hussain MT et al, Freeze-drying cycle optimization for the rapid preservation of protein-loaded liposomal formulations. International Journal of Pharmaceutics, p573, 118722, 2020.