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PHOTOLYTIC LABELING TO PROBE PEPTIDE-MATRIX INTERACTIONS IN LYOPHILIZED SOLIDSYuan Chen (5929574) 25 June 2020 (has links)
<p>Therapeutic proteins are often lyophilized with excipients such as sucrose or trehalose to protect them during manufacturing and achieve a longer shelf-life. Formulation design for therapeutic proteins has been a trial-and-error process, and the mechanisms responsible for the stabilizing effects of excipients are not fully understood. Two proposed theories have been widely accepted: the water replacement theory and the vitrification theory.<sup>1,2</sup>The water replacement theory suggests that excipients stabilize protein molecules in the solid state by forming hydrogen bonds that “replace” the hydrogen bonds to water that stabilize the protein in solution, while the vitrification theory asserts that proteins are stabilized by a glassy solid matrix of low mobility and does not require direct interactions between excipient and protein. A better understanding of the interactions between proteins and other components of the lyophilized matrix can facilitate rational formulation design and shorten the time in development. However, most of the analytical methods available can only provide information on the bulk properties of the lyophilized matrix such as moisture content and glass transition temperature (<i>T</i><sub>g</sub>); it has been difficult to measure the interactions between protein and excipient directly, if they exist. In order to characterize the interactions between protein and excipients in a lyophilized matrix with high resolution, a photolytic labeling method was developed in this dissertation, building on previous work in our research group. Photolytic labeling has long been used to identify protein-protein interactions <i>in vivo</i>.<sup>3,4</sup>Common types of photo-reaction reagents and their applications are summarized in Chapter 1. The research described in this dissertation utilizes the diazirine functional group, which is activated after UV exposure and undergoes a free radical reaction to form covalent bonds with nearby molecules. The reaction can be used to identify the interactions between excipients and protein or peptide in a solid formulation. Previous studies in our lab have shown that photo-reaction can be applied to lyophilized solids to study protein-matrix properties and interactions in the solid.<sup>5,6</sup>This dissertation seeks to further identify photo-reaction products and analyze them in a more quantitative way. </p><p> </p><p>Chapter 2 describes a quantitative analysis of photo-reaction products in solution and lyophilized solids using a model peptide, KLQ (Ac-QELHKLQ-NHCH<sub>3</sub>). The purpose of the work in this chapter is to establish a quantitative analytical method for photo-reaction products, enabling studies of peptide-excipient interactions in lyophilized solids. KLQ was derivatized with a bifunctional probe NHS-diazirine (succinimidyl 4,4’-azipentanoate; SDA) at Lys5 to be photo-reactive. The SDA derivatized KLQ (KLQ-SDA) was used to study the photo-reaction products and examine excipient interactions. Identification and quantitation of photo-reaction products of KLQ-SDA was achieved with liquid chromatography mass spectrometry (LC-MS) and reversed phase HPLC (rp-HPLC). Important reaction products such as peptide-excipient adducts and peptide water adducts varied in different formulations. Unexpected reaction products such as unproductive “dead-end” products and peptide-phosphate adducts from buffer salt were also detected and quantified. Together, the photo-reaction products reflected the local environment near Lys5 of the peptide in the solid state. This study has provided a better understanding of photo-reaction with diazirine in the lyophilized solids together with a quantitative description of the local environment near Lys5. </p><p> </p><p>In Chapter 3, the photo-reaction products in lyophilized solids exposed to increasing moisture were analyzed, and the effect of increasing moisture on the local environment near the peptide was examined. Using the analytical method developed in Chapter 2, these studies explored whether peptide-water interactions, as measured by the formation of water adducts formed by photolytic labeling, are linearly correlated with an increase in solid bulk moisture content. Formulations containing the KLQ-SDA peptide were exposed to various relative humidity conditions and photolytic labeling was induced. Solids containing disaccharide excipients behaved differently from those containing amino acids when exposed to the same relative humidity condition, showing different levels of peptide-excipient and peptide-water adducts. With increasing moisture content in the solids, the formation of photo-reaction products did not mimic the pattern of solutions with same composition, indicating differences in the local environment. </p><p> </p><p>An alternative approach to studying lyophilized formulations using photolytic labeling is to incorporate photo-reactive excipients into the solid matrix. In Chapter 4, a new diazirine-labeled photo-excipient, photo-glucosamine (pGlcN), was chemically synthesized and incorporated into formulations of the therapeutic peptide salmon calcitonin (sCT) and compared with the commercially available diazirine-labeled amino acid, photo-leucine (pLeu). The studies in Chapter 4 further compared peptide-excipient interactions at the molecular level with two different photo-excipients, ionizable pLeu and unionizable pGlcN. Changing solution pH prior to lyophilization was expected to change ionic interactions between sCT and pLeu in the solid samples, resulting in different distributions of photo-reactions products; pH-dependent differences were not expected for pGlcN. The results demonstrated that the distribution of photo-reaction products varied with the composition of the formulation and the pH of the solution prior to lyophilization. The photo-reaction products in the pGlcN-containing formulation differed from those pLeu, showing a difference in the interactions of unionizable (pGlcN) and ionizable (pLeu) excipients with sCT in solid samples. </p><p> </p><p>The work in this dissertation has developed photolytic labeling as a tool to study lyophilized peptide formulations, and has provided a more quantitative understanding of the photo-reaction products that are produced from diazirine-labeled peptides or excipients in the solid state. A new photo-reactive excipient has also been presented (pGlcN), which showed different photo-reaction products than a commercially available photo-excipient (pLeu) and is promising for future study. Photolytic labeling for formulation development is still in its early stages, and additional research regarding reaction mechanism and complementary stability studies is needed. Nevertheless, the results presented in this dissertation support continued development of photolytic labeling as a practical method for formulation design and development. </p><p> </p>
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Essays on the relationship between fertility and child mortality / Essais sur la relation entre mortalité infantile et féconditéBousmah, Marwân-al-Qays 05 February 2015 (has links)
Cette thèse se donne pour objectif de contribuer à la compréhension des tendances démographiques en Afrique subsaharienne par l’examen de l’influence de la mortalité infantile sur les comportements de fécondité. Dans le premier chapitre, j’examine la relation entre mortalité infantile et fécondité à l’échelle micro-économique. Des modèles de données de comptage sont utilisés pour analyser les déterminants de la fécondité complète de femmes d’une communauté rurale sénégalaise. Je montre que l’effet global de la mortalité infantile est positif tant sur la fécondité totale que sur la fécondité nette. De plus, j’identifie une relation en U inversé entre mortalité infantile et fécondité nette. Dans le second chapitre, j’analyse les effets de la mortalité infantile sur les comportements reproductifs dans un modèle de fécondité endogène où la survie infantile est stochastique. J’adopte une forme fonctionnelle de coût des enfants englobant quatre scénarios différents, chacun représentant un contexte socio-économique distinct. Mon modèle peut prédire des réponses positives et négatives de la fécondité, selon que les enfants sont respectivement “intensifs en temps” ou “pourvoyeurs en temps”. Finalement, le troisième chapitre analyse les effets de la mortalité et de la morbidité infantiles sur le processus de décision de fécondité des femmes rurales sénégalaises. J’estime des modèles dynamiques non linéaires de données de panel. Je montre que la mortalité et la morbidité palustres à l’échelle de la communauté, ont un effet positif sur les décisions ultérieures de fécondité. Cet effet est d’autant plus fort que la maladie est létale pour les enfants infectés. / This dissertation attempts to contribute to the understanding of current demographic trends in sub-Saharan Africa by examining the role of child mortality in shaping fertility behavior. In the first chapter of this dissertation, I examine the relationship between child mortality and fertility at the micro level. Count data models are employed to investigate the determinants of completed fertility of women from a Senegalese rural community. The global effect of child mortality on total and net fertility is found to be positive. I also identify an inverted-U shaped relationship between child mortality and net fertility. In the second chapter of this dissertation, I analyze the effects of child mortality changes on fertility behaviors in an endogenous fertility model where child survival is stochastic. I adopt a functional form for the cost of children that allows for four different scenarios, each of which is representative of a particular socio-economic setting. My model can predict both positive and negative fertility responses to child mortality depending on whether children are “time-intensive” or “time-supplying”, respectively. Finally, the third chapter analyzes the effects of childhood mortality and morbidity on the fertility decision-making process among rural Senegalese women. I estimate nonlinear dynamic panel data models of fertility behavior. I find that community child mortality and morbidity attributable to malaria exert a joint influence on fertility behaviors. Community-level malaria incidence among children has a positive effect on subsequent fertility choices, and this positive effect is stronger the more the disease is fatal to children who are infected.
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