Made available in DSpace on 2014-10-09T12:53:36Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:08:07Z (GMT). No. of bitstreams: 0 / Neste trabalho foi desenvolvida pela primeira vez uma estratégia de purificação com duas etapas, uma cromatografia de troca iônica e uma cromatografia líquida de alta eficiência em fase-reversa (RP-HPLC), para obter um hTSH derivado de CHO (r-hTSH-IPEN), que mostrou-se rápida e prática, permitindo um rendimento de 70% e uma pureza > 99%. Um aumento de ~60% na produtividade de r-hTSH-IPEN foi observado quando condições de cultura celular foram alteradas de 5% de CO2 para ar (0,03% CO2). A qualidade dos produtos obtidos em ambas as condições foi avaliada com relação à estrutura dos N-glicanos, aos isômeros de carga e à atividade biológica em comparação com a única preparação comercial conhecida (Thyrogen®) e com uma preparação de referência de origem hipofisária (p-hTSH) do National Hormone and Pituitary Program (NIDDK, EUA). Os N-glicanos identificados nas preparações recombinantes foram do tipo complexo, apresentando estruturas bi-, tri- e tetra- antenárias, algumas fucosiladas, sendo 86-88% sialiladas com níveis variáveis. As três estruturas mais abundantes foram monosialiladas, representando ~69% de todas as formas identificadas nas três preparações. A principal diferença foi encontrada em termos de antenaridade, com 8-10% mais estruturas bi-antenárias na ausência de CO2 e 7-9% mais estruturas tri-antenárias na presença de CO2. No caso do p-hTSH foram identificadas estruturas do tipo complexo, com alta-manose e híbridas, a maioria delas contendo resíduos terminais de ácido siálico e/ou sulfato. As duas estruturas mais abundantes contem um ou dois resíduos de sulfato, sendo que no primeiro caso ele inesperadamente se liga a uma galactose. A porcentagem de ligação do ácido siálico à galactose nas conformações 2-3 e 2-6 foi de 68 ± 10% e 32 ± 10% respectivamente. Não foram observadas diferenças fundamentais nos isômeros de carga, nas três preparações recombinantes, os perfis da focalização isoelétrica mostrando seis bandas distintas no intervalo de pI de 5,39 a 7,35. Uma distribuição consideravelmente diferente, com várias formas na região ácida, foi observada, no entanto, para duas preparações hipofisárias. Uma bioatividade ligeiramente superior (p <0,02) foi encontrada para o r-hTSH-IPEN obtido na presença de CO2 quando as preparações foram analisadas com boa precisão por um simples bioensaio em dose única; esta atividade, no entanto, não é significativamente diferente da atividade do Thyrogen, as duas preparações sendo 1,6 e 1,8 vezes mais potentes do que a preparação de referência (p-hTSH). Podemos concluir que, pelo menos para o caso dos r-hTSH derivados de CHO, diferentes condições de cultivo não afetam significativamente as estruturas dos N-glicanos, distribuição de isómeros de carga ou atividade biológica. Thyrogen e r-hTSH-IPEN, quando comparados com p-hTSH-NIDDK, apresentaram cerca de 7% de aumento da massa molecular determinada por MALDI-TOF-MS. Esta técnica, que permite uma avaliação exata da massa do heterodímero, apresentou valores de MR de 29611, 29839 e 27829, respectivamente. Diferenças significativas foram encontradas entre o r-hTSH e o p-hTSH pelo que se refere às propriedades hidrofóbicas, avaliadas por RP-HPLC. Também foram observadas diferenças relacionadas à composição de carboidratos, principalmente de ácido siálico e galactose, tendo sido encontrado um menor teor destes resíduos no p-hTSH. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:43:39Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:09:47Z (GMT). No. of bitstreams: 1 06652.pdf: 3582362 bytes, checksum: acb423f59896fb445118557871bc22b3 (MD5) / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Orientador: Cesar Costapinto Santana / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-19T00:34:49Z (GMT). No. of bitstreams: 1 Sousa_AnayladosSantos_D.pdf: 1255579 bytes, checksum: 2c0a223e27891ba584d4f621ffa8e2bd (MD5) Previous issue date: 2011 / Resumo: O Ácido hialurônico (AH) é um polissacarídeo linear com importantes aplicações na indústria farmacêutica, médica e cosmética, fazendo-se necessária sua purificação por ser oriundo de uma variedade de fontes, animal e não animal. Assim sendo, o presente trabalho discorre sobre a separação do AH produzido por fermentação em meio de cultura sintético, utilizando a técnica cromatográfica de exclusão por tamanho. Determinaram-se as melhores condições de separação utilizando, inicialmente, uma coluna de exclusão por tamanho operando em batelada. Estas condições foram mantidas em todos os ensaios e serviram de modelo para a obtenção dos parâmetros para o escalonamento do processo. Ainda em batelada, avaliou-se a inserção da técnica cromatográfica de troca iônica acoplada à exclusão por tamanho, com o intuito de aprimorar a separação do AH, averiguando resinas de caráter catiônico e aniônico. Realizou-se um estudo comparativo entre o processo em batelada, com e sem a inserção da troca iônica, e o semicontínuo operado com reciclo externo estacionário (REE), visando a obtenção de frações de AH e proteínas com elevado grau de pureza. Considerando-se o processo semicontínuo operado em REE foram avaliados diferentes volumes de reciclo, sendo eles 1,0; 1,5; 2,25 e 3,0 mL. Os resultados obtidos com o processo semicontínuo em REE alcançaram um percentual de purificação entre 92% e 97% para o AH, trabalhando-se com volumes de reciclo de 1,0 a 3,0 mL, enquanto que para as proteínas mostrou-se inviável. A inserção da cromatografia de troca iônica mostrou-se promissora com o uso da resina catiônica, apresentando 98% de pureza com um rendimento de 99% para o AH; ao contrário da resina aniônica que exibiu uma perda mássica de AH em torno de 50%. O escalonamento do processo cromatográfico de exclusão por tamanho mostrou-se possível, apresentando resultados semelhantes à operação em batelada / Abstract: The hyaluronic acid (HA) is a linear polysaccharide with important applications in the pharmaceutical, medical and cosmetics industry. Its purification is necessary because it is in a mixture coming from a variety of sources, animal and non-animal. The aim of this work is the separation of HA obtained by fermentation in synthetic medium, using the technique of size exclusion chromatography. The best conditions for HA separation were determined using, initially, a batch operation size exclusion column. These conditions were maintained in all trials and served to obtain the parameters for the large scale process. The batch operation also evaluated the ion exchange chromatography technique coupled to size exclusion, in order to enhance the separation of HA by examining cationic and anionic resins. A comparative study was carried out between the batch process, with and without the inclusion of ion exchange, and the semi-batch process with steady state recycling (SSR), in order to obtain fractions of HA and proteins with high purity. The semi-batch process operated with SSR assessed in differents volumes of recycle (1.0, 1.5, 2.25 and 3.0 mL). The results obtained with the semi-batch process with SSR achieved a purification percentage between 92 % and 97% for HA, working with recycle volumes from 1.0 to 3.0 mL, showing to be feasible for HA, while for the proteins proved to be unfeasible, with a maximum of 60 % purity. The ion-exchange chromatography obtained good results with the use of cationic resin, with 98% of purity and a yield of 99 % for HA, unlike the anionic resin which exhibited a mass loss of HA around 50 %. The scale-up process for size exclusion chromatography proved to be possible, with similar results to batch operation / Doutorado / Desenvolvimento de Processos Biotecnologicos / Doutor em Engenharia Química

M.Sc. / The behaviour i.e. the distribution, bioavailability and toxicity of an element depends on the particular species in which it is present. That being said the prediction of the behaviour of species can therefore not be predicted by determination of the total concentration alone. Speciation analysis is thus an important process, which seeks to identify and quantify the species that occur in a sample. By identifying the species one is better equipped to predict the behaviour of the element if it were released into the ecosystem as well as if it were used in industrial processes. The speciation analysis of the dominant chromium species, Cr(III) and Cr(VI), has received much attention as these contradictory species are involved in many industrial processes such as plating and tanning for example. The species have opposing behaviour as Cr(III) is found to be a necessary micronutrient for mammals where as Cr(VI) is known to be toxic. Of the numerous methods developed for chromium speciation analysis, the on-line process of chromatographic preconcentration followed by simultaneous detection with a sensitive detector produces a synergistic effect for the successful speciation analysis of chromium. It was thus decided to study ion chromatography hyphenated with an ICP-OES as a means of studying chromium species. Anion chromatography coupled with ICP-OES was the basis for the development of the first method. Complexation of Cr(III) with EDTA to form a monovalent anion preceded its separation from Cr(VI) as the chromate anion. A carbonate-bicarbonate eluent was used to elute the species from a Dionex AG 9 guard column coupled with an AS 9 separation column. This method proved to be effective and fast, with each analysis taking less than two minutes. The second method was based on cation chromatography in the form of a Dionex CG12A guard column being coupled with an ICP-OES spectrometer as the detector. This method made use of a gradient elution program in which hexavalent chromium was eluted first with ammonium nitrate followed by trivalent chromium with nitric acid. This method was successfully applied to synthetic samples and was found to be effective.

M.Sc. / The strontium isotope ratio 87Sr/86Sr in the wine and soil of four wine-producing regions in South Africa has been determined using quadrupole ICP-MS. Isotope ratio measurements with fairly good precision had been obtained and 87Sr/86Sr ratio values in wine and soil samples were statistically evaluated to determine whether the regions investigated could be distinguished. Statistical interpretation of data revealed that the Robertson wine-producing region could be distinguished from Stellenbosch, Swartland and Walker Bay wine regions on the basis of their 87Sr/86Sr ratio. By using a combination of 87Sr/86Sr and multielement data, canonical discriminant functions with very good classification abilities could be obtained to correctly distinguish between wine and soil samples originating from a specific production region. Due to the isobaric overlap of strontium and rubidium at mass 87, a very good strontium-rubidium separation method had to be developed. The chelating properties of EDTA and DCTA in combination with ion exchange chromatography were explored in order to achieve such a separation. Due to the advanced rubidium separation ability of the developed DCTA elution method, it has been employed in all wine and soil sample preparations. Since chromatographic ion exchange procedures are not capable of removing all the rubidium from the sample matrices, 87Sr/86Sr ratio values were mathematically corrected for residual rubidium using an 88Sr correction. Since strontium isotope ratio measurements require very good precision, it is traditionally determined using TIMS. However, due to the ease of operation, shorter analysis time and widespread availability of quadrupole ICP-MS, the technique was evaluated for its merit for determining strontium isotope ratios. The optimization of acquisition parameters for the determination of the 87Sr/86Sr ratio in wine and soil samples with good precision of below 0.1% RSD has been conducted. Furthermore, selected sample matrix components were assessed as possible matrix interferences as well as for their ability to affect the precision of measurements. It was concluded that with adequate optimization of acquisition parameters, proper sample preparation and reliable mathematical correction of data, 87Sr/86Sr ratio measurements with reasonable precision could be obtained using quadrupole ICP-MS.

>Magister Scientiae - MSc / 68Ge/68Ga generators rely on metal oxide, inorganic and organic sorbents in order to prepare radionuclides useful for clinical applications. The requirements for 68Ge/68Ga generators are that the 68Ga obtained from the 68Ge loaded column should be optimally suited for the routine synthesis of 68Ga-labelled radiopharmaceuticals, that the separation of the 68Ga daughter from the 68Ge parent should happen easily, with a high yield of separation, a low specific volume of 68Ga and should not contain trace elements owing to the solubility of the metal oxide sorbent. Beginning with a metal oxide preparation and continuing through recent developments, several approaches for processing generator derived 68Ga have altered the production of 68Ge/68Ga generators. Still, the effects of sorbent modification on the properties of 68Ge/68Ga radionuclide generator systems are not necessarily optimally designed for direct application in a medical context. The objective of this research was to analyze and document characteristics of Titanium Oxide (TiO2) sorbents relevant to processing of a 68Ge/68Ga generator that is able to produce 68Ga eluates that are adequate for clinical requirements. Interest was shown in TiO2 based 68Ge/68Ga generators by a number of overseas companies for tumour imaging using 68Ga-labelled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated peptides. While a method involving production of the 68Ga radionuclide using TiO2 metal oxide had been published, problems with the production persisted. A method, using TiO2 metal oxide for ion exchange chromatography, was devised in this study to produce the 68Ga radionuclide, with the aim of being adopted for production purposes. The study focuses on the development of a dedicated procedure for the achievement of sufficient 68Ga yield along with low 68Ge breakthrough and low metallic impurities. Literature from 1970 to 2011 was reviewed to assess the radiochemical aspects of the 68Ga production and processing thereof. Various commercially available TiO2 metal oxides were characterized by subjecting the materials to x-ray diffraction (XRD), x-ray fluorescence (XRF) and scanning electron microscopy (SEM) for quantitative and qualitative analysis.

Fluoride ion is an accompanying impurity in a wide variety of chemical gypsum throughout the world. In this study, the Ion Selective Electrode (ISE) method, the Ion Chromatography (IC) method and the standard Willard and Winter method of fluoride analysis were adapted and compared for use in the quantification of fluoride in South African chemical gypsum. During the use of ISE, the pH of sample solutions was found to be a critical parameter for the results to be meaningful. An operating pH of approximately 5 was suitable for consistency of results. It was important to ensure the existence of the ionised form of fluorine in solution, because the detection was based on the sensitivity of the membrane electrode to this species. In the case of the Willard and Winter method, the traditional visual indicator titration was replaced by a more sensitive spectrophotometric detection, because of low fluoride levels in the chemical gypsum. The parameter sensitive reaction rate approach was adapted, and the reaction allowed to go to completion to enable measurement with a bench top spectrophotometer. The IC method required a good separator since fluoride ions usually eluted too early for detection on common ion exchange columns. The data handling of the chromatographic software was thoroughly examined and consistent integration of the chromatograms maintained. Sample preparation of the chemical gypsum involved particle size reduction through grinding. No trend between fluoride impurity and the particle size of the sample was observed. <p. The quantity of fluoride in Kynoch and Omnia phosphogypsum were found to be 0.10% and 0.04% respectively. The Tioxide chemical gypsum, titangypsum, was found to contain in the region of 0.02% fluoride. Generally, the level of fluorine (F) has to be reduced to about 0.2% before phosphogypsum can be used as substitute for natural gypsum in the cement industry. The aim of this study is to critically evaluate three analytical methods, namely, the Willard and Winter standard method of fluoride analysis, the ISE method, and IC as applied in the quantification of fluoride in chemical gypsum. Secondly, the efficiency of treatment of the chemical gypsum with water, lime and sulphuric acid was investigated. The ISE method was found to be faster and relatively simpler versus both Willard and Winter and the IC methods. The IC method was quite superior for indicating general complexity of the sample and it was faster than the Willard and Winter method. The standard Willard and Winter method was generally found to be long and tedious. The three methods validated one another as percentages of fluoride in the chemical gypsum samples were the same. The study indicated that a sulphuric acid pretreatment of the chemical gypsum was the most effective (90%) in the removal of fluoride impurity compared to the lime treatment and water washing (9%). However, the water washing would be inexpensive for practical treatment of chemical gypsum at plant level. / Thesis (PhD(Chemistry))--University of Pretoria, 2006. / Chemistry / unrestricted

<p>  </p> <p>As monoclonal antibodies (mAbs) become a more important part of the pharmaceutical industry, the need for better quicker analysis of then will also increase. To do this, better stationary phases specifically designed for mAbs need to be developed to analyze the quality of mAbs by their critical control attributes. The three main critical control attributes are size, charge, and glycosylation. This work focuses mainly on the development of stationary phases to analyze critical control attributes in size and charge through using a non-porous silica base and surface confined atom transfer radical polymerization to grow improved stationary phases that minimize undesired interactions and maximize desired interactions.</p>

Monoclonal antibodies are large, complex biomolecules that can be difficult to characterize. Characterization is important because of the various post translational modifications that can occur during manufacturing, processing, and storage. Some modifications can lead to efficacy and safety issues and therefore are heavily monitored. A leading way to monitor various modifications is by using liquid chromatography. The high sensitivity, reproducibility, and ability to quantitate analytes makes it very attractive for monoclonal antibody characterization. The large molecular size of monoclonal antibodies (150 kDa) makes them challenging to separate efficiently and with high enough resolution to be helpful. New column technologies that would help improve protein separation efficiencies and slectivities would greatly help in this challenging process. In this thesis, three novel bonded phases are developed for the separation of monoclonal antibodies including a weak anion and cation exchanger (WAX, CEX) for the separation of charged species as well as a novel hydrophilic interaction chromatography (HILIC) for the separation of glycoforms. Column develop is achieved by optimizing selectivity and improving efficiency of separations by altering particle surface chemistry.

It is the purpose of this, then, to investigate the feasibility of using the changes of concentrations of small iconic decomposition products in bile as indicators of time since death. High pressure liquid chromatography was selected for measurement of these changes.