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Title: The Development of Novel Combined Thermal and Imaging Approaches to the Characterisation of Frozen Trehalose Solutions
Author: Wu, Jiejun
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2008
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Abstract:
The purpose of this thesis is to investigate the properties of trehalose solutions as cryoprotectants for biopharmaceuticals with respect to the glass transition, relaxation behaviour and protective function. A novel combined approach using MTDSC, cryo AFM and DMA was employed to characterise the frozen trehalose solution in terms of its glass transition temperature. The observations made are consistent with a model in which there are crystalline regions between which there is an amorphous connecting matrix. A substantial amount of the amorphous phase softens at the temperature of the first transition (Tnl) representing the glass transition of this phase. Associated with this transition is crystallisation which can be seen as an exotherm in the non-reversing signal, manifested by an increase in crystal size and an increase in complex modulus. As the temperature increases toward the temperature region of the second transition (Tr2), the volume of crystals decreases in an inhomogeneous manner before finally vanishing. This indicates that the second transition must be related to the start of the loss of crystallinity or the melting. Moreover, the freeze concentration (CO for frozen trehalose solution was calculated by a new proposed enthalpy integration method with baseline correction. This method shows good agreement with the commonly used method involving calculation from the melting endotherm of ice. However, the new method avoids the uncertainties that are caused by the different integral baseline construction and indicates an improved experimental accuracy in the results. The calculated freeze concentration of non-annealed and annealed (20 hours) trehalose solution are 73.1% and 79.2% respectively (the mole ratio of unfrozen water and trehalose changes from 7: 1 to 5: 1). A new approach combining two mathematical models, the Lacey-Reading-Nickolopolous and Gordon-Taylor equations, are introduced to explain the decrease in heat capacity during this annealing. It suggests that this is due to the glass transition temperature shifting to higher temperatures, caused by the recrystallisation of unfrozen water in the amorphous phase. A trend of changes with the mole ratio of water and trehalose for the different concentration trehalose solutions during the annealing process are also successfully plotted as a function of time. Furthermore, the dielectric response of different concentration frozen trehalose solutions were examined and the relaxation time was calculated by curve fitting the peak maximum of imaginary permittivity into the Dissado-Hill model. The apparent activation energy was calculated via the Arrhenius equation and the results indicate that the dielectric relaxation in the trehalose frozen solution is not dominated by ice or the hydroxyl group and the hydroxymethyl side groups in the sugar; the manner of relaxation response in such system is mostly associated with unfrozen water. In addition, a more complex system containing a model protein lactate dehydrogenase (LDH) and trehalose as cryoprotectant is studied under the freeze stresses. FT Raman and biological assay are the methods that use to check the integrity of the protein structure. The data show the perturbation of the secondary structure especially from the wave range of Amide I in Raman spectrum and loss of activity of the protein after the freeze-thawing process. The low concentration LDH only solutions showed more damage to the secondary structure of the protein and less recovered activity after freezing. However, for solutions to which trehalose was added, the well preserved Amide I peaks in FT Raman results and the better maintained activity data suggested that little perturbation happened to the native structure of LDH after frozen.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.514319  DOI: Not available
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