Presently, over 300 proteins or peptide based therapeutic medicines have been approved by the FDA owing to advances in protein engineering and technology. However, majority of these protein-based medications are unstable or have limited shelf life when in aqueous form. During pre-formulation and manufacturing, various technological processes including mixing, dissolving, filling (through pipes) can produce strong mechanical stresses on proteins. These stresses may cause the protein molecule to unfold, denature or aggregate. To improve stability upon formulation, they may be manufactured as freeze dried cakes that requires reconstitution with a buffer or water prior to administration. Although it has been successful in improving the stability of protein-based formulations, the freeze drying process itself also contributes to protein aggregation. This process introduces other stresses such as freezing, thawing and drying. In addition to these stresses, the agitation processes used during reconstitution may also destabilize the protein’s native structure. Two key processes used in preparation of protein based formulations were studied in this work; mechanical agitation and freeze drying. The aim of this project was to explore the aggregation of proteins that occur due to the various technological processes typical in the production of protein based formulations. The project has two parts that relates to liquid and solid formulations. In the first part, the effect of different methods of mechanical agitations on BSA protein was investigated. In the second part, the focus was on the effect of formulation (i.e. the application of amino acids) on aggregation of protein (BSA) in freeze dried formulations. Arginine and lysine were added individually into protein-based freeze-dried formulation to study their potential of improving the stability of the proteins during manufacturing, storage and reconstitution. In the formulation development, additional excipients were added to prevent moisture uptake due to the hygroscopic properties of the amino acids and to provide lyo- and cryo- protection for the protein molecule during freeze drying. Without further purification, BSA solutions prepared by using sonication, low shear rotor mixer or high shear tube/pipe mixing were studied using dynamic light scattering (DLS). Thioflavin T assay and turbidimetry analysis were used as complementary studies. In protein-based freeze dried formulations, at accelerated storage conditions, the presence of aggregates were studied in samples containing arginine or lysine using ThT assay and turbidimetry analysis. Characterisation of the freeze dried cakes was performed relative to their moisture sorption, cake shrinkage, mechanical properties and morphology using various analytical techniques. iv In the BSA solution studies, particle size analysis indicated two distributions for non-agitated BSA solution that corresponds to the average particle sizes of BSA molecules and their aggregates. Under mechanical stresses (all types), the intensity of distribution centered ≈ 7.8 nm reduces and broadens as the agitation time increases, indicating a reduction in the amount of “free” BSA macromolecules. The second distribution, as a result of increasing agitation time or shear intensity, reveals a significant shift towards larger sizes, or even splits into two particle size populations. These particle size growths reflect the formation of aggregates due to intensive collisions and, as a result, partial unfolding followed by hydrophobic interactions of exposed non-polar amino acids. UV spectra showed that aggregation in both low shear and mechanical vibration agitations were lower compared to the high shear stress. When compared to non-agitated BSA solution, ThT assay recorded ≈15 times higher fluorescence emission from the high shear samples, ≈2 times fluorescence emission from low shear and ≈6 times fluorescence emission from mechanical vibrations. Thus all the three agitation methods showed a good correlation between the results. The second part of this project was performed in three stages. In the initial 2 stages, 2- and 3-excipients component system were investigated to develop an optimal preliminary formulations which will be used in the final protein based 4-components formulations. From the 1st stage (ArgHCl/LysHCl + sugar/polyol), among 4 tested excipients (polyol and sugar), mannitol was observed to have resisted moisture uptake by the highly hygroscopic ArgHCl/LysHCl amino acids. However, mannitol is considered a good cryoprotector but has poor lyoprotection properties. Therefore, in the following stage, a 3rd excipient (in a 3-excipients component system) sucrose or trehalose, was introduced into the formulation. The formulation was made up of 20% ArgHCl (LysHCl), and various ratios of mannitol and sugar were explored. The criteria for selecting the best systems were based on ideal physicochemical properties i.e. moisture uptake, shrinkage, mechanical properties, matrix structure and appearance, and thermal properties. The final stage was the formulation of a 4-components system comprising the three excipients and combinations selected from the stage 2 studies, and the addition of BSA as the model protein. To study aggregation in this system, a freeze dried 4-components excipient/protein system was reconstituted and incubated at accelerated storage conditions over time. Fluorescence spectroscopy and turbidimetry were used to study aggregation of proteins, moisture uptake kinetics with gravimetric balance, and thermal analytical techniques were used to characterise the freeze dried cakes with and without BSA protein. This study represented a systematic analysis of aggregation of proteins in both liquid and solid formulations. Some of the novel aspects of this study include: v 1. The new experimental results obtained for aggregation of proteins in solution subjected to mechanical agitations. The high shear stress created by syringe agitation, simulated the real situation in post manufacturing process during filling through narrow pipes, and has been shown here to strongly affect the aggregation of protein macromolecules. 2. The development of a methodical approach for optimization of multi component (up to 4 excipients) protein based formulations. 3. The unexpected non-linear behavior of the physicochemical properties of the 3-excipients component system as a function of composition. To the best of my knowledge, this novel aspect has not been previously reported in literature. 4. Application of amino acid in protein based formulations has shown the inhibition of aggregation of BSA, with the highest effect observed with ArgHCl. The results of this study coincide with the conclusions published previously for aggregation of proteins in solution.

The in origin intertidal marine brown alga Fucus vesiculosus L. grow permanently sublittoral in the brackish Bothnian Sea, side by side with the recently discovered F. radicans L. Bergström et L. Kautsky. Environmental conditions like salinity, light and temperature are clearly different between F. vesiculosus growth sites in the Bothnian Sea (4-5 practical salinity units, psu; part of the Baltic Sea) and the tidal Norwegian Sea (34-35 psu; part of the Atlantic Ocean). The general aims of this thesis were to compare physiological aspects between the marine ecotype and the brackish ecotype of F. vesiculosus as well as between the two Bothnian Sea species F. vesiculosus and F. radicans. The result in the study indicates a higher number of water soluble organic compounds in the marine ecotype of F. vesiculosus compared to the brackish ecotype. These compounds are suggested to be compatible solutes and be due to an intertidal and sublittoral adaptation, respectively; where the intertidal ecotype needs the compounds as a protection from oxygen radicals produced during high irradiation at low tide. The sublittoral ecotype might have lost the ability to synthesize these compound/compounds due to its habitat adaptation. The mannitol content is also higher in the marine ecotype compared to the brackish ecotype of F. vesiculosus and this is suggested to be due to both higher level of irradiance and higher salinity at the growth site. 77 K fluorescence emission spectra and immunoblotting of D1 and PsaA proteins indicate that both ecotypes of F. vesiculosus as well as F. radicans have an uneven ratio of photosystem II/photosystem I (PSII/PSI) with an overweight of PSI. The fluorescence emission spectrum of the Bothnian Sea ecotype of F. vesiculosus however, indicates a larger light-harvesting antenna of PSII compared to the marine ecotype of F. vesiculosus and F. radicans. Distinct differences in 77 K fluorescence emission spectra between the Bothnian Sea ecotype of F. vesiculosus and F. radicans confirm that this is a reliable method to use to separate these species. The marine ecotype of F. vesiculosus has a higher photosynthetic maximum (Pmax) compared to the brackish ecotype of F. vesiculosus and F. radicans whereas both the brackish species have similar Pmax. A reason for higher Pmax in the marine ecotype of F. vesiculosus compared to F. radicans is the greater relative amount of ribulose-1.5-bisphosphate carboxylase/oxygenase (Rubisco). The reason for higher Pmax in marine ecotype of F. vesiculosus compare to the brackish ecotype however is not due to the relative amount of Rubisco and further studies of the rate of CO2 fixation by Rubisco is recommended. Treatments of the brackish ecotype of F. vesiculosus in higher salinity than the Bothnian Sea natural water indicate that the most favourable salinity for high Pmax is 10 psu, followed by 20 psu. One part of the explanation to a high Pmax in 10 psu is a greater relative amount of PsaA protein in algae treated in 10 psu. The reason for greater amount of PsaA might be that the algae need to produce more ATP, and are able to have a higher flow of cyclic electron transport around PSI to serve a higher rate of CO2 fixation by Rubisco. However, studies of the rate of CO2 fixation by Rubisco in algae treated in similar salinities as in present study are recommended to confirm this theory. / Fucus vesiculosus L. (Blåstång) är en brunalg som i huvudsak växer i tidvattenzonen i marint vatten men arten klarar också att växa konstant under ytan i det bräckta Bottenhavet. Norska havet och den del av Bottenhavet, där algerna är insamlade i denna studie, har salthalterna 34-35 psu (praktisk salthaltsenhet) respektive 4-5 psu. F. radicans L. Bergström et L. Kautsky (Smaltång) är en nyligen upptäckt art (2005) som har utvecklats i Bottenhavet. F. radicans och Bottenhavets ekotyp av F. vesiculosus växer sida vid sida och har tidigare ansetts vara samma art. Sett till hela Östersjön, så ändras ytans salthalt från 25 till 1-2 psu mellan Östersjöns gräns mot Kattegatt och norra Bottenviken. Den låga salthalten i Östersjön beror på det höga flödet av sötvatten från älvarna och på ett litet inflödet av saltvatten i inloppet vid Kattegatt. Salthaltsgradienten är korrelerad med antalet arter som minskar med minskad salthalt. Östersjön är ett artfattigt hav och de arter som finns är till stor del en blandning av söt- och saltvattenarter. Det finns bara ett fåtal arter som är helt anpassade till bräckt vatten och F. radicans är en av dem. Exempel på miljöskillnader för F. vesiculosus i Norska havet och i Bottenhavet är salthalten, tidvattnet, ljuset och temperaturen. Tidvattnet i Norska havet gör att algerna växlar mellan att vara i vattnet och på land, vilket utsätter algerna för stora ljusskillnader, snabba och stora temperaturväxlingar samt även torka. De alger som växer i Bottenhavet har däremot en jämnare och lägre temperatur, istäcke på vintern och mindre tillgång på ljus eftersom de alltid lever under vattenytan. Skillnaderna i miljön mellan växtplatserna leder till skillnader i fysiologiska anpassningar. Anledningen till att F. vesiculosus och F. radicans valdes som studieobjekt i denna avhandling är att de är viktiga nyckelarter i Bottenhavet. F. vesiculosus och F. radicans är de enda större bältesbildande alger som finns i det artfattiga ekosystemet och de används därför flitigt som mat, gömställe, parningsplats och barnkammare för t.ex. fisk. Att de är nyckelarter gör det angeläget att försöka förstå hur algerna är anpassade och hur de reagerar på miljöförändringar för att få veta hur de kan skyddas och bevaras. F. radicans inkluderades även för att se hur en naturlig art i Bottenhavet är anpassad i jämförelse med den invandrade F. vesiculosus. Marin F. vesiculosus inkluderades för att vara en artreferens från artens naturliga växtplats. Studien visar att det finns fler vattenlösliga organiska substanser (finns vissa organiska substanser som har en proteinskyddande funktion) i den marina ekotypen av of F. vesiculosus än i Bottenhavets ekotyp. Anledningen till detta föreslås vara en anpassning till att växa i tidvattenzonen. Vid lågvatten utsätts F. vesiculosus från Norska havet för starkt ljus, uttorkning, och snabba temperatur- växlingar vilket gör att den kan behöva dessa organiska substanser som skydd mot fria syreradikaler som bildas under lågvattenexponeringarna. F. vesiculosus från Bottenhavet har troligen mist förmågan att syntetisera dessa substanser på grund av anpassning till att hela tiden växa under ytan. Mängden mannitol (socker) är högre i den marina ekotypen av of F. vesiculosus än i Bottenhavets ekotyp. Detta föreslås bero på högre fotosyntetiskt maximum i F. vesiculosus från Norska havet jämfört med ekotypen från Bottenhavet. Skillnaden i fotssyntetiskt maximum är bland annat kopplat till ljus- och salthaltskillnaden på algernas växtplatser. Denna teori styrks av att både fotosyntesen och halten av mannitol ökar i Bottenhavets ekotyp när den behandlas i högre salthalt. Studien visar även att båda ekotyperna av F. vesiculosus samt F. radicans har ett ojämnt förhållande mellan fotosystem II och I (PSII och PSI) med en dominans av PSI. Denna slutsats är baserad på fluorescens emissions mätningar vid 77 K (-196 °C) och mätning av den relativa mängden D1 protein (motsvarar PSII) och PsaA protein (motsvarar PSI). F. vesiculosus från Bottenhavet visar ett emission spektrum som pekar mot en jämnare fördelning av PSII och PSI jämfört med den marina ekotypen och F. radicans. Detta stämmer dock inte med förhållandet mellan D1/PsaA som indikerar att alla tre har mer PSI än PSII. Förklaringen till avvikelsen mellan metoderna antas vara att F. vesiculosus från Bottenhavet har större ljus-infångande antennpigment än marin F. vesiculosus och F. radicans. De tydliga skillnaderna i 77 K fluorescens emission spektra mellan Bottenhavets F. vesiculosus och F. radicans visar att denna metod kan användas som säker artidentifiering. Den marina ekotypen av F. vesiculosus har högre fotosyntetiskt maximum än de båda arterna från Bottenhavet. Mätningar av den relativa mängden av enzymet Rubisco, viktigt för upptaget av koldioxid hos växter och alger, visar att mängden enzym är en sannolik förklaring till skillnaden i fotosyntetiskt maximum mellan den marina ekotypen av F. vesiculosus och F. radicans och detta är troligen en normal artskillnad. Mängden Rubisco kan dock inte förklara skillnaden i fotosyntetiskt maximum mellan de båda ekotyperna av F. vesiculosus. För att undersöka vad skillnaden mellan dessa två beror på så föreslås istället mätningar av Rubisco’s koldioxidfixeringshastighet. Det är en ökning av fotosyntetiskt maximum i Bottenhavets ekotyp av F. vesiculosus när den behandlas i högre salthalt (10, 20 och 35 psu) och det högsta fotosyntetiska maximumet uppmättes i alger som behandlats i 10 psu. Denna ökning beror inte på ökning i den relativa mängden av Rubisco. Ökningen i fotosyntesen speglas dock av en ökning av den relativa mängden PsaA. Detta antas bero på att det behövs mer energi i form av ATP och att en ökning av detta kan ske på grund av att mer PsaA kan driva den cykliska elektrontransporten i fotosyntesreaktionen. Ökat behov av ATP antas bero på en ökning av Rubisco aktiviteten men mätning av aktiviteten krävs för att bekräfta detta.

Roll compaction is a continuous process commonly used in the pharmaceutical industry for dry granulation of moisture and heat sensitive powder blends. It is intended to increase bulk density and improve flowability. Roll compaction is a complex process that depends on many factors, such as feed powder properties, processing conditions and system layout. Some of the variability in the process remains unexplained. Accordingly, modeling tools are needed to understand the properties and the interrelations between raw materials, process parameters and the quality of the product. It is important to look at the whole manufacturing chain from raw materials to tablet properties. The main objective of this thesis was to investigate the impact of raw materials, process parameters and system design variations on the quality of intermediate and final roll compaction products, as well as their interrelations. In order to do so, we have conducted a series of systematic experimental studies and utilized chemometric tools, such as design of experiments, latent variable models (i.e. PCA, OPLS and O2PLS) as well as mechanistic models based on the rolling theory of granular solids developed by Johanson (1965). More specifically, we have developed a modeling approach to elucidate the influence of different brittle filler qualities of mannitol and dicalcium phosphate and their physical properties (i.e. flowability, particle size and compactability) on intermediate and final product quality. This approach allows the possibility of introducing new fillers without additional experiments, provided that they are within the previously mapped design space. Additionally, this approach is generic and could be extended beyond fillers. Furthermore, in contrast to many other materials, the results revealed that some qualities of the investigated fillers demonstrated improved compactability following roll compaction. In one study, we identified the design space for a roll compaction process using a risk-based approach. The influence of process parameters (i.e. roll force, roll speed, roll gap and milling screen size) on different ribbon, granule and tablet properties was evaluated. In another study, we demonstrated the significant added value of the combination of near-infrared chemical imaging, texture analysis and multivariate methods in the quality assessment of the intermediate and final roll compaction products. Finally, we have also studied the roll compaction of an intermediate drug load formulation at different scales and using roll compactors with different feed screw mechanisms (i.e. horizontal and vertical). The horizontal feed screw roll compactor was also equipped with an instrumented roll technology allowing the measurement of normal stress on ribbon. Ribbon porosity was primarily found to be a function of normal stress, exhibiting a quadratic relationship. A similar quadratic relationship was also observed between roll force and ribbon porosity of the vertically fed roll compactor. A combination of design of experiments, latent variable and mechanistic models led to a better understanding of the critical process parameters and showed that scale up/transfer between equipment is feasible.

Abdominal surgery inhibits gastrointestinal motility, a phenomenon referred to as postoperative ileus. Since the postoperative ileus disturbs duodenal physiology it is important to minimize the side effects of this condition. Recent experiments in our laboratory show that treatment of anaesthetised rats with parecoxib, a selective cyclooxygenase-2 inhibitor, prevents duodenal postoperative ileus, increases duodenal mucosal bicarbonate secretion and improves other functions as well. One aim of the thesis was to investigate whether removal of luminal chloride affect the parecoxib- and the vasoactive intestinal peptide (VIP)-induced stimulation of duodenal mucosal bicarbonate secretion. The proximal duodenum of anaesthetised Dark Agouti rats was perfused with isotonic solutions containing zero or low Cl- and the effect on luminal alkalinisation determined. The basal as well as the parecoxib-induced increase in alkalinisation, but not that stimulated by VIP, were markedly reduced in the absence of luminal Cl-. One important function of the duodenum is to adjust luminal osmolality towards that in the blood. It is believed that the adjustment of osmolality in the duodenum is achieved by osmosis and diffusion of electrolytes along their concentration gradients and that these processes occur predominately paracellularly. Another aim of the thesis was to examine whether prevention of postoperative ileus affects the duodenal response to luminal hypertonicity. The proximal duodenum of anaesthetised Dark Agouti and Sprague-Dawley rats were perfused with hypertonic solutions of different composition and osmolality and the effects on duodenal motility, alkaline secretion, transepithelial fluid flux, mucosal permeability and the adjustment of luminal osmolality were determined in absence and presence of parecoxib. It is concluded that COX-2 inhibition increases duodenal mucosal bicarbonate secretion by stimulating apical Cl-/HCO3- exchange in duodenocytes. Furthermore, pretreatment of anaesthetised rats with parecoxib improves a number of duodenal functions in both rat strains that contribute to improve the ability to adjust luminal osmolality. The choice of rat strain is another important feature to consider when interpreting the results because the DA strain was more responsive to luminal hypertonicity than the SD strain. Finally, several evidences are provided to suggest that the adjustment of luminal osmolality in the rat duodenum is a regulated process.

Amoebae of the genus Acanthamoeba spp. are free-living unicellular organisms found in disparate ecosystems all over the world. Due to their ability to invade human body, evade its defensive mechanisms and cause extensive tissue damage, Acanthamoeba infection can lead to serious, if rare, diseases, affecting most commonly the eye and the central nervous system. Specific therapy for Acanthamoeba infections is not available. A major reason for therapeutic failure in ameobiasis is the ability of the protist to differentiate into resistant stages. These are cysts, known to be formed under prolonged unfavorable conditions, both in the environment and the infected tissues, and the pseudocysts, less durable but rapidly formed under acute stress. The present thesis focuses on as yet unexplored mechanisms of resistance of cysts and pseudocysts. Moreover, further characteristics distinguishing cysts and pseudocysts as well as the processes involved in their formation are investigated. One of the issues addressed is a presence of protective carbohydrate compounds mannitol and trehalose that participate in defensive reactions against abiotic stress in many organisms. Although putative genes for enzymes of the trehalose and mannitol synthetic pathways are present in the genome of Acanthamoeba, only one of the...

Doctor of Philosophy (PhD) / Asthma is a chronic inflammatory disease of the airways. Corticosteroid medication is the most effective currently available treatment. Complications of corticosteroid therapy are dose-dependent, however, the clinical efficacy of varying doses of inhaled corticosteroids has been studied with mixed results. A randomized, double-blind, parallel group study was used to evaluate the inhaled corticosteroid dose-response relationship for clinical endpoints and in vitro parameters of underlying airway inflammation and remodelling. The mannitol provocation test with Forced Oscillation Technique (FOT) was used to derive potential dose-differentiating endpoints. In vitro inflammatory markers were measured in alveolar macrophages from bronchoalveolar lavage. Basement membrane thickness was measured from bronchial biopsies. Eleven nonasthmatic subjects were enrolled for comparison. This thesis addresses the null hypothesis that there is no significant difference in clinical and biological effects between low dose (200mcg/day, n=11) and high dose (1000mcg/day, n=11) treatment (for 6-7 weeks) with inhaled fluticasone propionate (FP) for a range of clinical outcomes and in vitro markers of airway inflammation and remodelling. Significant changes after FP included increased FEV1, reduced airway hyperresponsiveness (AHR) (by FOT and FEV1), exhaled nitric oxide and Juniper symptom score. In addition, significant reductions occurred in expression of GM-CSF, TNF-alpha and IL-1ra in macrophages. A lower baseline FOT-derived respiratory system conductance was predictive of a greater degree of improvement in symptoms. No statistically significant differences in the changes after treatment between low and high dose FP were found in spirometry, exhaled nitric oxide, symptom scores, AHR, alveolar macrophage cytokine levels (GM-CSF, TNF-alpha, IL-1ra, IL-10) and basement membrane thickness, although there were trends towards greater improvements in many of the parameters after high dose FP. Basement membrane thickness appeared to be reduced by high dose FP, although this reduction was not statistically significant. There was a weak, but statistically significant, negative correlation between basement membrane thickness and FOT-derived conductance (r2=0.135, p=0.042). With the recognition of the limitations in the interpretation of these data, the results suggest that, in previously steroid naïve mild to moderate asthmatics, there may be only minimal benefit derived from an additional 800µg/day of inhaled fluticasone above the low dose of 200µg/day.

Ce mémoire est consacré à la mécanosynthèse d'alliages moléculaires par cobroyage. Les composés étudiés sont des composés utilisés comme excipients dans l'industrie pharmaceutique. Il s'agit plus particulièrement du lactose, du tréhalose et du mannitol. Nos investigations ont montré la possibilité d'obtenir, sur une certaine gamme de concentrations, des solutions solides vitreuses homogènes tréhalose/mannitol et lactose/mannitol par cobroyage des poudres cristallines correspondantes. Elles ont aussi permis de montrer de manière originale l'influence de la position relative de la température de broyage par rapport à la température de transition vitreuse (Tg) sur la nature des états obtenus par broyage en faisant varier la zone de Tg des alliages par changement de composition chimique. Les transformations sous broyage des composés purs ont également été étudiées en détail. Le broyage du lactose et du tréhalose en dessous de Tg est à l'origine d'une transformation directe cristal -> verre à l'état solide. Par contre, le broyage des formes cristallines d et ß? du mannitol au dessus de Tg engendre une transformation polymorphique vers la forme cristalline a de stabilité intermédiaire montrant, de ce fait, le caractère stationnaire des états atteints au cours de ce type de transformations. De plus, nos investigations ont permis de déterminer un certain nombre de propriétés spécifiques de ces systèmes inaccessibles à partir des liquides purs et des mélanges liquides. La mutarotation du lactose a pu être caractérisée en détail. Les diagrammes de phases des mélanges lactose/mannitol et tréhalose/mannitol ont pu être établis. L'ensemble de ces résultats a été obtenu par DRX, RMN, DSC et ATG.

During photosynthesis Plantaginaceae species can produce glucose derivatives such as iridoid glycosides and alcohol sugars that in addition to sucrose can be exported from leaves. Plantago lanceolata transported sorbitol in addition to sucrose especially at warmer leaf temperatures. However, two iridoids, catalpol and aucubin, found in P. lanceolata were not readily labelled from 14CO2 under any conditions examined. In contrast, in two greenhouse, cut-flower cultivars of Antirrhinum majus the iridoids, antirrhinoside and antirrhide, were readily 14C-labelled along with sucrose but little 14C was recovered in alcohol sugars (e.g., mannitol). The amount of 14C-partitioned into antirrhinoside increased at higher temperatures. Exposing leaves of P. lanceolata and A. majus to reduced-photorespiratory conditions (e.g. short-term CO2 enrichment and/or low O2) increased fixation and export. Under low O2 in P. lanceolata sorbitol 14C-labelling increased relative to sucrose and in A. majus 14C-labelling of sucrose increased relative to antirrhinoside. Also 14C-labelling of antirrhide increased more than antirrhinoside. During both short-term and long-term acclimation to high CO2, whole plant NCER, leaf photosynthesis and export increased in A. majus. Taken together the temperature and CO2 enrichment studies show plasticity in Plantaginaceae species to synthesize and transport sucrose and auxiliary glucose esters and alcohol sugars in a species-specific manner (depending on the rate of carboxylation).

This dissertation has focused on studying the effect of four glycine derivatives on the solid state properties of mannitol, glycine, and sucrose when freeze dried into blended mixtures. The primary goal was to assess their value for use in the stabilisation of vaccines in the solid state, by examining key physical and chemical characteristics, which have been documented to be beneficial to the stabilisation of biopharmaceutical formulations. The novel excipients; dimethyl glycine, and trimethyl glycine, were shown to retard the crystallisation and increase the overall glass transition temperature, of mannitol, when freeze dried as evidenced by DSC and Powder X-ray diffraction. Mannitol’s glass transition temperature increased from 100C to 12.650C and 13.610C when mixed with methyl-glycine and dimethyl glycine respectively. The glycine derivatives did not show the same effect on sucrose which remained amorphous regardless of the concentration of the other excipient. The different behaviour with the sucrose system was thought to be due to relatively high glass transition temperature of sucrose. Conversely glycine remained highly crystalline due it’s relatively low glass transition temperature. The novel excipient formulations were also assessed for their effect on the aggregation of the adjuvant aluminium hydroxide when freeze dried by Dynamic Light Scattering (DLS).The formulations containing the glycine derivatives all caused a decrease in the aggregation size of the adjuvant from ~26 μm, to 185 nm in the presence of methyl glycine. The effects of lysozyme and viral antigen on the adjuvants were also examined showing that the addition of the virus did not affect the size of the aggregates formed, however lysozyme showed significant decreases in the aggregates formed. Examination of the freezing method were also made showing that faster freezing rates produced smaller aggregates of the adjuvant. When investigating the rate at which the excipients lost water during secondary drying there was evidence of the formation of hydrates of glycine, trimethyl glycine, and mannitol has shown that the glycine derivatives have attributes which would be beneficial in stabilising vaccines in the solid state when freeze dried.

This dissertation has focused on studying the effect of four glycine derivatives on the solid state properties of mannitol, glycine, and sucrose when freeze dried into blended mixtures. The primary goal was to assess their value for use in the stabilisation of vaccines in the solid state, by examining key physical and chemical characteristics, which have been documented to be beneficial to the stabilisation of biopharmaceutical formulations. The novel excipients; dimethyl glycine, and trimethyl glycine, were shown to retard the crystallisation and increase the overall glass transition temperature, of mannitol, when freeze dried as evidenced by DSC and Powder X-ray diffraction. Mannitol’s glass transition temperature increased from 100C to 12.650C and 13.610C when mixed with methyl-glycine and dimethyl glycine respectively. The glycine derivatives did not show the same effect on sucrose which remained amorphous regardless of the concentration of the other excipient. The different behaviour with the sucrose system was thought to be due to relatively high glass transition temperature of sucrose. Conversely glycine remained highly crystalline due it’s relatively low glass transition temperature. The novel excipient formulations were also assessed for their effect on the aggregation of the adjuvant aluminium hydroxide when freeze dried by Dynamic Light Scattering (DLS).The formulations containing the glycine derivatives all caused a decrease in the aggregation size of the adjuvant from ~26 μm, to 185 nm in the presence of methyl glycine. The effects of lysozyme and viral antigen on the adjuvants were also examined showing that the addition of the virus did not affect the size of the aggregates formed, however lysozyme showed significant decreases in the aggregates formed. Examination of the freezing method were also made showing that faster freezing rates produced smaller aggregates of the adjuvant. When investigating the rate at which the excipients lost water during secondary drying there was evidence of the formation of hydrates of glycine, trimethyl glycine, and mannitol has shown that the glycine derivatives have attributes which would be beneficial in stabilising vaccines in the solid state when freeze dried. / Stabilitech Ltd. and the Engineering and Physical Sciences Research Council (EPSRC).