A NOVEL DIELECTRIC TECHNIQUE FOR MONITORING THE LYOPHILISATION OF GLOBULAR PROTEINS
Background. The moisture content of lyophilised proteins plays an important role in the stability and long-term storage of these substances. Residual moisture is mainly determined by the combination of formulation factors and the cycle-times of the various stages of lyophilisation. The final drying stage of the lyophilisation process, i.e. secondary drying, is a critical stage in determining the optimum residual moisture level. Some measurement techniques have been developed for in situ monitoring of moisture content during secondary drying. However, all these techniques involved the direct contact of a measurement probe with the freeze-dried material, which may alter the properties of the finished product. There is a challenge, therefore, to establish a non-destructive method for in situ monitoring of water content and the determination of the end point of the lyophilisation process. Aim. The framework of this study was to develop a dielectric technique that is able to monitor water content of a material inside a glass vial. The technique was based on the development of a remote-electrode system, that avoids the introduction of measurement probes into the sample. In the context of this work, a remote electrode system is defined as electrodes that are separated from the sample by a non-conductive and non-dispersive medium, i.e. the glass vial used for the freeze drying process. As an initial development, this study dealt with dielectric measurements on partially hydrated globular proteins, i.e. ovalbumin, lysozyme, and pepsin. Method. The development of the new technique is necessarily based on a detailed knowledge of the dielectric properties of water-protein interactions. The first stage of the project therefore, involved a thorough study of the low frequency dielectric properties of hydrated proteins using conventional parallel plate electrodes. The next stage involved the simulation of a simple remote electrode measurement, by placing non-conductive, non-dispersive polymer films between the sample and parallel plate electrodes. Finally, the study on dielectric measurement of hydrated proteins contained in a glass vial was undertaken with the electrodes attached to the glass vial. Other physical measurements such as FTIR, SEM, XRD, and DSC, were also employed as complementary techniques to characterise hydrated and freeze-dried protein samples. Results. The investigation of the dielectric behaviour of selected hydrated proteins showed two independent mechanisms of proton hoppings in the bulk sample, which were distinctly identified as an anomalous low frequency dispersion and a dielectric loss peak (3 dispersion). An understanding of the polarisation process underpinning the dispersion is based on the cluster model, in which the low frequency dispersion (LFD) is associated with proton hopping between clusters of water molecules (inter-cluster transport), and the 3 dispersion is associated with proton hopping between water molecules in the same cluster (intra-cluster transport). Protons were shown to be the charge carriers involved in both dispersions by investigating the dielectric behaviour of proteins hydrated with deuterated and normal water. Both the LFD and 3 dispersion moved simultaneously toward high frequency with the increase of temperature or hydration. At a certain temperature, however, both dispersions shifted back to lower frequency. This phenomena was identified as a percolation threshold. In the hydration study, the percolation threshold was recognised as critical hydration. This critical hydration was considered due to the monolayer of water molecules network covering a single macromolecule protein, therefore creating an infinite cluster, or known as percolation threshold. The critical hydration was also observed from both the calculation of fractal dimension (Df) and the activation energy (H) of the Arrhenius behaviour at below percolation threshold. For all selected proteins, it was found that below critical hydration level, the proton transport occurred along the surface of protein macromolecule (Df < 1.9); whereas above critical hydration level, the proton transport may occur within the matrix of protein structure (Df > 1.9). With the application of the remote electrode system, it was found that the dielectric properties for the 3 dispersion was unaffected by the insertion of polyethylene films between the sample and the conventional parallel plate electrodes. The dielectric properties for the 3 dispersion was also unaffected in measurement using remote electrode system in the form of custom-made electrodes attached externally to the glass vial. Moreover, the 3 dispersion was still sensitive to the water content, irrespective of which remote electrode system was used. A reasonable correlation (R2 = 93%) was also observed between relaxation times (3) obtained using conventional parallel plate electrodes and the remote electrodes attached to glass vial. Conclusion. The study has revealed the mechanism and interaction of water-protein for hydrated globular proteins. The study has also shown that dielectric measurements using remote electrodes, attached to a glass vial, are applicable for the in situ measurement of water content in materials, for example in the determination of the end point of the lyophilisation process.
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