103Pd is an important therapeutic radionuclide and has recently found great interest due to its higher radiobiologic effect. 109Cd decays by electron capture and is generally used as calibration sources in industrial and medical fields. A new method for the production of 103Pd and 109Cd using the 66 MeV proton beam of iThemba LABS on a tandem natural silver target (Ag/Ag) has been developed. The tandem targets (each target with a mass of 9 g and a thickness of 3 mm) were placed in the high energy slot (62.515 MeV - 40.173 MeV) and low energy slot (38.652 MeV – 0 MeV) to produce the bulk 103Pd and 109Cd, respectively. The radiochemical separation of the Pd radionuclides (103Pd, 100Pd) and the co-produced Rh radioisotopes (mainly 101Rh and 100Rh which are produced from decay of their Pd parents) from the bulk natAg was achieved using a Chelex chelating resin column. In the preliminary studies, different size columns (3 cm x 1 cm, 11 cm x 1 cm, 13 cm x 1 cm and 16 cm x 1.5 cm) were investigated to determine the optimal column conditions for the separation. It was determined that the optimal conditions for the chemical separation was with a 13 cm x 1 cm resin column with the elution of Rh and Ag radionuclides carried out with 1 M HNO3 and the elution of Pd radionuclides with 10 M HCl. No Ag or Rh impurities were detected in the final product and the average recovery of Pd was > 96 %. This work was repeated using a ―hot‖ irradiated Ag target and the chemical processing was done in a hot cell using the same resin column conditions. The recovery of the high purity 103Pd from the irradiated natAg target was found to be > 95 %. The radiochemical separation of 109Cd from the bulk natAg target was done in two parts. In the first part, the precipitation method was used to reduce the silver into a metallic form using 30 g of Cu turnings. The resulting 109Cd filtrate was loaded onto a AG-X10 anion exchange resin column (6 cm x 1 cm). For the optimal chemical separation, the elution of Ag and Cu(II) was carried out with 2 M HCl containing H2O2 and the elution of 109Cd was accomplished with 1 M HNO3. The recovery yield of 109Cd was > 99 %. / Chemistry / M.Sc. (Chemistry)

Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Radiochemical research involving ion exchange chromatography is of paramount importance to the future of radionuclide production at the Radionuclide Production Group (RPG) of iThemba LABS. It is required for the production of high-activity yields of radionuclides to effectively remove impurities and for the safety of the operators performing such productions. The radiochemical separations of some new products from their target material, as well as experiments to determine whether production is viable, are described. 67Ga is currently being produced at the RPG and makes use of zinc targets. With the production of ultra-pure 67Ga, it was necessary to remove any Fe(III) impurities from the final product, such that it may be possible to label peptides with this product. The use of Amberchrom CG161M for this purpose was found to be satisfactory. Interest was shown in 88Y by an overseas company for the manufacture of sources. While a method involving extraction of the radionuclide and the ion exchange thereof using Chelex 100 chelating resin had been published, problems with the production persisted. Three methods, using ion exchange chromatography, were devised to produce the radionuclide, with two of them being adopted for production purposes. Thick-target nuclear data have also recently been accumulated in collaboration with colleagues from ATOMKI, Debrecen, Hungary. There is a large demand for 82Sr for the manufacture of 82Sr/82Rb generators for medical use. A method was developed to manufacture this radionuclide with thicker (32 g) target material, bombarded in the Vertical Beam Target Station (VBTS), and to separate 82Sr from its target material with the use of Purolite S950 chelating resin. 68Ge/68Ga generators are becoming increasingly important in the world of radiopharmaceuticals. A project to develop a local generator was funded by the Innovation Fund and research was performed to produce 68Ge, such that the generator could be manufactured. This involved bombarding thicker Ga targets in the VBTS and performing the chemical separation using AG MP-1 anion exchange resin. The final product was loaded onto generators, although tests performed on different materials to the ones being marketed are also reported in this work. A project was initiated to study the cluster radioactive decay of 223Ac via 14C and 15N emission. To produce 223Ac for these observations, a Th target was bombarded. The 227Pa was separated from the target material using AG MP-1 macroporous anion exchange resin and used as a source, which decayed to 223Ac. The chemical separation and the drying of the final product onto a source plate were completed within approximately 70 minutes from the end of bombardment. The work was performed in collaboration with JINR, Russia, and University of Milan and INFN, Italy. 133Ba has a half-life of over 10 years and is an expensive radionuclide to produce. It has been used in medical and biological studies and there still appears to be a demand for it. A method was devised, utilizing AG50W-X4 cation exchange resin, to separate 133Ba from its CsCl target material. Agricultural specialists in the past have shown an interest in 28Mg, to determine the uptake of the element in fruit. It has long been regarded by some of the local researchers as an interesting project to investigate. It has been determined that the product can be produced in reasonable quantities using LiCl target material, with ten targets being bombarded in series using a 200 MeV proton beam delivered by the Separated Sector Cyclotron. A method, involving the use of Purolite S950 chelating resin, was devised to separate 28Mg from its target material. / AFRIKAANSE OPSOMMING: Radiochemiese navorsing, wat ioonuitruiling chromatografie behels, is van uiterste belang vir die toekoms van die produksie van radionukliede by die Radionukliedproduksiegroep (RPG) van iThemba LABS. Dit is nodig vir die hoë aktiwiteit opbrengs van radionuklied produkte om onsuiwerhede te verwyder en vir die veiligheid van die operateurs wat die produksies moet uitvoer. Die skeiding van nuwe produkte van hulle skyfmateriaal, sowel as eksperimente om vas te stel of ‘n produksie uitvoerbaar is, word in die werk beskryf. 67Ga word tans by RPG vervaardig en maak gebruik van sink as skyfmateriaal. Vir die produksie van “ultra-suiwer” 67Ga was dit belangrik om enige Fe(III) onsuiwerhede uit die finale produk te verwyder om sodoende peptiede merking te kan uitvoer. Die gebruik van Amberchrom CG161M hars was voldoende vir dié eksperiment. ‘n Oorsese maatskappy het belangstelling getoon in 88Y vir die vervaardiging van bronne. Alhoewel ‘n metode wat die ekstraksie van die radionuklied en die ioonuitruiling daarvan met die gebruik van Chelex 100 chelerende hars reeds gepubliseer was, het probleme met die produksie voortgeduur. Drie metodes is opgestel om 88Y te produseer, waarvan twee van die metodes tans gebruik word vir produksie doeleindes. Dik-skyf kerndata is ook versamel in samewerking met kollegas van ATOMKI, Debrecen, Hongarye. Daar is ‘n groot aanvraag vir 82Sr vir die vervaardiging van 82Sr/82Rb generators vir mediese doeleindes. ‘n Metode is ontwikkel om die radionuklied te vervaardig van dikker skyfmateriaal (32 g), in die Vertikale Bundelstasie gebombardeer, en om 82Sr van sy skyfmateriaal te skei met die gebruik van Purolite S950 chelerende hars. 68Ge/68Ga generators is besig om toenemend belangrik te word in die wêreld van radiofarmasie. iThemba LABS kry baie navrae om die produk te vervaardig. Die projek was ook deel van die voorlegging aan die “Innovation Fund” en ‘n manier is ondersoek om 68Ge te vervaardig, wat benodig word om so ‘n generator te laai. Dik Ga skyfmateriaal word in die Vertikale Bundelstasie gebombardeer en ‘n chemiese skeiding is uitgevoer deur gebruik te maak van AG MP-1 anioonuitruiling hars. Die finale produk is op die generators gelaai vir toetsdoeleindes. Toetse is ook op ‘n ander tipe generator uitgevoer en word in die werk beskryf. ‘n Projek is begin om “cluster” radioaktiewe verval van 223Ac, via 14C en 15N emissie, te bestudeer. ‘n Th-skyf is met protone gebombardeer om die 223Ac te produseer vir die eksperiment. 227Pa is vervaardig en geskei van die skyfmateriaal. Dit is gedoen met die gebruik van AG MP-1 makroporeuse anioonuitruiling hars en drooggemaak op ‘n bronplaat, waar dit verval het na 223Ac. Die chemiese skeiding en die droogmaak van die finale produk op ‘n bronplaat is uitgevoer binne 70 minute na Einde van Bombardering (EVB). Die werk is deel van ‘n samewerking met kollegas van JINR, Rusland, en die Universiteit van Milaan, sowel as INFN, Italië. 133Ba het ‘n halveertyd van oor die tien jaar en is ‘n duur produk om te vervaardig. Dit is al gebruik in mediese en biologiese studies en daar is deesdae ‘n redelike aanvraag daarvoor. ‘n Metode is uitgewerk om 133Ba te skei van die CsCl skyfmateriaal met die gebruik van AG50W-X4 katioonuitruiling hars. Spesialiste in landboustudies het in die verlede belangstelling getoon in 28Mg. Dit word gebruik om die absorpsie van dié element in vrugte te ondersoek. Die produk kan vervaardig word met die gebruik van LiCl skyfmateriaal: tot soveel as tien skywe (agter mekaar) word gebombardeer met ‘n 200 MeV protonbundel te iThemba LABS. ‘n Metode, wat Purolite S950 behels, is daargestel om 28Mg van die skyfmateriaal te skei.

The objective of this research is to further define the relationship between the charge state of insulin, and the self assembly properties of insulin and PEGylated insulin in solution. Polyethylene glycol (PEG) chains were covalently attached to insulin in order to evaluate their impact on insulin's systemic duration of action after pulmonary dosing. This thesis will focus on the assembly properties of the PEG-insulin and insulin, and also demonstrate how the charge state, which was modified by the covalent attachment of PEG, relates to different modes of behavior by anion and cation exchange chromatography. In addition, explain how modifying the assembly state extends to improving formulation properties of spray-dried insulin powders. This thesis is an investigation into the relationship of insulin's charge state controlled by pH and how the charge state affects the self assembly of insulin, especially when the zinc ion is removed. Ionic interaction is one of the major forces affecting insulin assembly. The theory that a change in the charge state of insulin could modulate the ionic interaction and reduce hexamer formation at alkaline conditions was investigated. Experiments were designed to measure the level of hexamer with light scattering, and the amount of hexamer was then correlated with the pH and zinc content of the solutions. The importance of the charge state of the monomer and its behavior extends to chromatography and purification modes as well. Specifically, the purification of various species of PEGylated insulin presents a challenge. By varying mobile phase pH which induces the charge to insulin, an ion exchange method demonstrated very high resolution and controllable interaction between the ion exchange media and the insulin derivatives. A highly accurate method for determining molecular weight and thus the average associated state of insulin in solution has been developed using the MALS (Multi-Angle Light Scattering). Insulin concentration, pH, and metal ion concentrations, were in pharmaceutically relevant ranges. The MALS method was developed to evaluate how the parameters above affect the self-assembly properties of insulin, and use the assembly properties to improve formulations of insulin or PEGylated insulin. To use the light scattering technique the dn/dc (change in refractive index with change in concentration) is required. During the method development, the dn/dc of insulin was measured at 690 nm, and a value of 0.185 mL/g based on theory was confirmed. A novel approach for preparing insulin powders with improved chemical stability, based on maintaining the dissociation of hexamers in solution during the spray drying process was developed. The mode presented here is to remove the zinc ions from solution, increase the pH from 6.6 to 7.8, and maintain a low concentration of insulin approximately 2 to 15 mg/mL. Each of these factors alone decreases the hexamer population in solution, but by combining all three factors, hexamers are driven to very low levels of equilibrium. The increased stability of the powders is predominately related to the decrease in covalent insulin dimer (CID). The data presented correlates a reduced hexamer population in the solution with lower levels of CID's in the dry powder compared to controls. The CID formation rate was reduced by 40% compared to a control.

Previous studies in Dr. Yang’s laboratory have established a grafting, design, and subdomain approach in order to investigate the properties behind Ca2+-binding sites located in Ca2+-binding proteins by employing engineered proteins. These approaches have not only enabled us to isolate Ca2+-binding sites and obtain their Ca2+-binding affinities, but also to investigate conformational changes and cooperativity effects upon Ca2+ binding. The focus of my thesis pertains to optimizing the expression and purification of engineered proteins with tailored functions. Proteins were expressed in E. coli using different cell strains, vectors, temperatures, and inducer concentrations. After rigorous expression optimization procedures, proteins were further purified using chromatographic and/or refolding techniques. Expression and purification optimization of proteins is essential for further analyses, since the techniques used for these studies require high protein concentrations and purity. Evaluated proteins had yields between 5-70 mg/L and purities of 80-90% as confirmed by SDS-PAGE electrophoresis.

Facile procedures based on hyphenated ion-exchange chromatography (IEC) and inductively coupled plasma mass spectrometry (ICP-MS) were developed to determine conditional stability constants, speciate chromium species and investigate the reduction of Cr(VI). 1. Improvements were made to a method previously developed to determine the conditional stability constant, Kf’, and chelation number, n, using IEC-ICP-MS. This method allowed the accurate determination of the conditional stability constant of a simple system. However, the corresponding chelation number was significantly different to the expected value because the principal assumption, i.e. that the ligand was in excess, was not realized in the experimentation. Furthermore, it neglected complexes other than that formed with EDTA4-. By taking into account these factors, accurate Kf’ and n were obtained for Co-EDTA and Zn-EDTA systems. 2. A simple method was developed for chromium speciation analysis at sub-µg L-1 level in potable water by IEC-ICP-MS. Cr(VI) and Cr(III) were separated on IonPac® AG-7 guard column within 7.5 minutes using gradient elution with 0.1 M ammonium nitrate and 0.8 M nitric acid. H2 collision/reaction interface gas eliminated chlorine-based and carbon-based polyatomic interferences on Cr detection. Water samples were analyzed directly, without any pretreatment. The accuracy of the method was verified through accurate analysis of riverine water certified reference material. Limits of detection of 0.02 and 0.04 µg L-1 for Cr(VI) and Cr(III), respectively, were obtained. 3. This speciation analysis method was then used for kinetics studies of Cr(VI) reduction in acidified riverine water. Water was spiked with Cr(VI), with or without Cr(III), and evolution of each Cr species with time was monitored by speciation analysis, showing that the reduction of Cr(VI) was a pseudo first order reaction. By plotting the logarithm of the peak area ratio of the instant Cr(VI) concentration over that of the original spiking versus time, the reaction rate constant was obtained as the slope. The reduction rate increased with decreasing pH and increasing temperature. The activation energy of the reaction at pH 1.3 was calculated using an Arrhenius plot. This method offers the advantages of small sample consumption, minimal sample manipulation, and easy data interpretation. / Thesis (Ph.D, Chemistry) -- Queen's University, 2010-09-30 08:05:27.342

Baculoviruses are versatile viruses that can be used as biopestisides, or for the production of recombinant protein and vaccines. Baculoviruses have also been found to be able to transfer genes to mammalian cells. This finding opened the door for the application of baculovirus vectors in human gene therapy. However, the mass production of clinical grade baculovirus vectors is challenging. Downstream processing has now become the bottle-neck of the manufacturing process. In this work, an anion exchange chromatography-based process was investigated for the purification of recombinant baculovirus vectors using a novel hydrogel based membrane (Natrix Separations Ltd.). Crude recombinant baculovirus supernatant from infected insect cell cultures was first subjected to a clarification process consisting of centrifugation and filtration. The pH of the viral solution was adjusted and then passed through a fast protein liquid chromatography system consisting of the ion exchange membrane. After washing weakly bound impurities, the captured baculoviruses are recovered by an elution step. Overall, baculoviruses strongly associated with the membrane; however, this interaction which was much physical as it was chemical, could not be entirely reversed and baculovirus was lost in the process. Product purity has also been evaluated and up to 85% of total protein reduction was determined. The significant losses of baculovirus observed have indicated major limitations in using this membrane for the purification of baculovirus.

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1ABSTRACTCarbohydrate-deficient transferrin (CDT) is a biomarker for excessive and long-running intake of alcohol. It is a form of transferrin called disialotransferrin that under normal circumstances is <2 % of total transferrin in human blood. An increase is seen when alcohol consumption exceeds450 g per week. CDT is analyzed in serum usinghigh performance liquid chromatography (HPLC) and UV/VIS detection. The purpose of this study was to investigate if an “in routine” method could be improved by switching columns.With ion exchange chromatography transferrin glycoforms are separated and quantified. The carbohydrate-deficient transferrin glycoforms have an isoelectric point between 5,7-5,9 that depends on the number of sialic acids on the molecule. With the use of a salt gradient and pH above the isoelectric point the glycoforms can beseparated depending on their affinity to the stationary phase. Batches with patient and control serum was first analyzed on the routine column Source 15Q PE and then on the alternative column Reprospher 200 SAX 5μm.Student’s t-test showed that the two methods’results correlated but were significantly different. A Bland-Altman plot illustrated differences between the two columns. High and low control serum values from Reprospher were lower than the accepted interval. In this study Reprospher’s stationary phase seemed to be affected to such an extent that stabile retention time, better resolution, and stabile values could not be achieved and because the information about the column was lacking an attempt to regeneratethe columnwas not conducted.

Orientador: Luis Henrique Souza Guimarães / Banca: Ariela Veloso de Paula / Banca: Rosimeire Cristina Linhari Rodrigues Pietro / Banca: Hamilton Cabral / Banca: Sandra Regina Pombeiro Sponchiado / Resumo: Produtos naturais bioativos podem ser produzidos por organismos de todos os reinos, de procariotos a eucariotos. Entre esses, os fungos filamentosos são fontes abundantes de diversos metabólitos, como proteínas e enzimas, as quais vêm se destacando devido as suas diversas atividades biológicas, com aplicações não só na área farmacêutica e médica como também, nas indústrias alimentícia, de materiais e na agricultura. Recentemente, a resistência microbiana frente aos antibióticos disponíveis no mercado e o aparecimento de linhagens tumorais cada vez mais agressivas, torna urgente a investigação por novas moléculas bioativas, especialmente aquelas com baixa ou nenhuma toxicidade contra as células de mamíferos. Devido ao grande potencial das proteínas fúngicas, como promissoras moléculas com aplicações biotecnológicas, o objetivo deste projeto foi realizar a prospecção de proteínas bioativas secretadas por fungos filamentosos do gênero Aspergillus. Entre as diferentes espécies estudadas, Aspergillus niveus cultivado em fermentação submersa (FSbm), em meio YPD por 120 h, foi capaz de secretar uma proteína com massa molecular de 19 kDa. A proteína foi analisada por espectrometria de massas e os peptídeos identificados apresentaram identidade de 88 a 100% com outras ribotoxinas já descritas na literatura, como α-sarcina, restrictocina e Asp f1. A ribotoxina inibiu a proliferação celular in vitro de linhagens tumorais de glioblastoma (até 32%), melanoma (43%), osteossarcoma (41%) e m... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Natural products can be produced by organisms from all kingdoms, from prokaryotes to eukaryotes. Among these, filamentous fungi are abundant sources of various metabolites, such as proteins and enzymes, which have been outstanding due to their diverse biological activities, with applications not only in the pharmaceutical and medical areas but also in the materials and food industries and in agriculture. Recently, microbial resistance to available antibiotics and the increasingly of aggressive tumor cell lines, makes urgent the investigation of new bioactive molecules, especially those with low or without toxicity to mammalian cells. Due to the great potential of fungal proteins, as promising molecules with biotechnological applications, the objective of this project was the prospection of bioactive proteins secreted by filamentous fungi of the genus Aspergillus. Among the different species studied, Aspergillus niveus cultured under submerged fermentation (Sbmf), in YPD medium for 120 h, was able to secrete a protein with molecular mass of 19 kDa. The protein was analyzed by mass spectrometry and the peptides identified showed 88 to 100% of identity with other ribotoxins described in the literature, such as α-sarcin, restrictocin and Asp f1. The ribotoxin inhibited, in vitro, the cell proliferation of glioblastoma tumor cell line (up to 32%), melanoma (43%), osteosarcoma (41%) and medulloblastoma (50%) at concentration of 20 μg / mL and incubation for 72 h. The ribotoxin was ... (Complete abstract click electronic access below) / Doutor

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