The potential application of a hydrogel-based strong anion-exchange (Q) membrane to purify plasmid DNAs was evaluated. The maximum binding capacity of plasmid DNA was estimated to be 12.4 mg/ml of membrane volume with a plasmid DNA recovery of ~ 90%, which is superior to other commercially available anion-exchange resins and membranes. The membrane was able to retain its structural integrity and performance after multiple cycles of usage (> 30 cycles). The inherent properties of plasmid DNA, membrane adsorbent, and the ionic environment on membrane performance were identified as the factors affecting membrane performance and their effects were systematically investigated. Plasmid DNAs with smaller tertiary structure have shorter dynamic radius and/or lowersurface charge densities, which tended to have a better adsorption and recovery than those with larger tertiary structure. Environmental Scanning Electron Microscopy (ESEM) revealed that the hydrogel structure is more porous on one side of membrane than the other, and higher plasmid DNA adsorption and recovery capacities were observed if the more porous side of the membrane was installed upward of flow in the chromatographic unit. ESEM also revealed improved pore distribution and increased membrane porosity if membrane was pre-equilibrated in the buffer solution for 16 hours. The development of better flow through channel in the hydrogel membrane upon extensive soaking further improved plasmid DNA adsorption and recovery capacities. The ionic environment affects the tertiary size of plasmid DNA; and the optimal operating pH of membrane chromatography was different for the plasmid DNAs investigated in this study. The relative contribution of these factors to improve membrane chromatography of plasmid DNAs was analyzed using statistical modeling. It was found that the adsorption of plasmid DNA was mainly affected by the available adsorptive area associated with membrane porosity, whereas the recovery of plasmid DNAs was mainly affected by the environmental pH. A novel, RNase-free, and potentially scalable bioprocess was synthesized using the hydrogel membrane as the technology platform for the manufacturing of pharmaceutical-grade plasmid DNA. High bioprocess recovery and product quality were primarily associated with the optimal integration of impurity removal by calcium chloride precipitation and anion-exchange membrane chromatography and the implementation of isopropanol precipitation as a coupling step between the two impurity-removing steps. Complete removal of total cellular RNA impurity was demonstrated without the use of animal-derived RNase. High-molecular-weight (HMW) RNA and genomic DNA (gDNA) were removed by selective precipitation using calcium chloride at an optimal concentration. Complete removal of the remaining low-molecular-weight (LMW) RNA was achieved by membrane chromatography using the high-capacity and high-productive hydrogel membrane. The simultaneous achievement of desalting, concentrating and buffer exchange by the coupling step of isopropanol precipitation and the high efficiency and resolution of DNA-RNA separation by anion-exchange membrane chromatography significantly reduced the operating complexity of the overall bioprocess, increased the overall recovery of plasmid DNA, and enhanced product quality by removing trace amounts of impurities of major concern for biomedical applications, such as gDNA, proteins, and endotoxin.

The potential application of a hydrogel-based strong anion-exchange (Q) membrane to purify plasmid DNAs was evaluated. The maximum binding capacity of plasmid DNA was estimated to be 12.4 mg/ml of membrane volume with a plasmid DNA recovery of ~ 90%, which is superior to other commercially available anion-exchange resins and membranes. The membrane was able to retain its structural integrity and performance after multiple cycles of usage (> 30 cycles). The inherent properties of plasmid DNA, membrane adsorbent, and the ionic environment on membrane performance were identified as the factors affecting membrane performance and their effects were systematically investigated. Plasmid DNAs with smaller tertiary structure have shorter dynamic radius and/or lowersurface charge densities, which tended to have a better adsorption and recovery than those with larger tertiary structure. Environmental Scanning Electron Microscopy (ESEM) revealed that the hydrogel structure is more porous on one side of membrane than the other, and higher plasmid DNA adsorption and recovery capacities were observed if the more porous side of the membrane was installed upward of flow in the chromatographic unit. ESEM also revealed improved pore distribution and increased membrane porosity if membrane was pre-equilibrated in the buffer solution for 16 hours. The development of better flow through channel in the hydrogel membrane upon extensive soaking further improved plasmid DNA adsorption and recovery capacities. The ionic environment affects the tertiary size of plasmid DNA; and the optimal operating pH of membrane chromatography was different for the plasmid DNAs investigated in this study. The relative contribution of these factors to improve membrane chromatography of plasmid DNAs was analyzed using statistical modeling. It was found that the adsorption of plasmid DNA was mainly affected by the available adsorptive area associated with membrane porosity, whereas the recovery of plasmid DNAs was mainly affected by the environmental pH. A novel, RNase-free, and potentially scalable bioprocess was synthesized using the hydrogel membrane as the technology platform for the manufacturing of pharmaceutical-grade plasmid DNA. High bioprocess recovery and product quality were primarily associated with the optimal integration of impurity removal by calcium chloride precipitation and anion-exchange membrane chromatography and the implementation of isopropanol precipitation as a coupling step between the two impurity-removing steps. Complete removal of total cellular RNA impurity was demonstrated without the use of animal-derived RNase. High-molecular-weight (HMW) RNA and genomic DNA (gDNA) were removed by selective precipitation using calcium chloride at an optimal concentration. Complete removal of the remaining low-molecular-weight (LMW) RNA was achieved by membrane chromatography using the high-capacity and high-productive hydrogel membrane. The simultaneous achievement of desalting, concentrating and buffer exchange by the coupling step of isopropanol precipitation and the high efficiency and resolution of DNA-RNA separation by anion-exchange membrane chromatography significantly reduced the operating complexity of the overall bioprocess, increased the overall recovery of plasmid DNA, and enhanced product quality by removing trace amounts of impurities of major concern for biomedical applications, such as gDNA, proteins, and endotoxin.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A precipitaÃÃo de Ãons metÃlicos com sulfeto à um processo importante no tratamento Ãguas residuÃrias oriundas de efluentes industriais de acabamento metÃlico. Sobretudo, por ser uma alternativa viÃvel para a recuperaÃÃo e reutilizaÃÃo de metais jà que o controle de pH a torna seletiva. O presente trabalho estudou a precipitaÃÃo seletiva de Cu2+, Zn2+ e Ni2+ utilizando H2S(g) gerado pela hidrÃlise da Tioacetamida. Os estudos foram realizados em escala laboratorial em duas etapas. Na primeira etapa, foram realizados estudos monoelementar para cada Ãon (100 mg.L-1), sendo inicialmente investigado os seguintes parÃmetros: montagem do sistema reacional, faixa de pH (2,0 a 6,0) e tempo de exposiÃÃo ao gÃs H2S (de 30 a 150 minutos). O estudo mostrou que o Cu2+ precipita imediatamente quando à exposto ao H2S(g) e em qualquer valor de pH testados alcanÃando 99,9% de remoÃÃo. Para o Zn2+ a precipitaÃÃo foi preferencial quando o pH ficou entre 4,0 e 5,0, e o tempo de exposiÃÃo ideal foi de 60 min, para que a precipitaÃÃo fosse acima de 95,0%. O pH influenciou todo o processo de precipitaÃÃo dos Ãons, sendo mais influente para o Ni2+, iniciando sua precipitaÃÃo em pH 6,0, e apÃs 4 ciclos de 30 min, somando um tempo de exposiÃÃo de 120 min para que a sua remoÃÃo fosse acima de 90%. Na segunda etapa, foram realizados estudos de mistura binÃria e ternÃria dos Ãons a partir dos valores de pH e tempo de exposiÃÃo encontrados pelo estudo individual. Em resumo, os resultados obtidos nos experimentos feitos com a mistura dos Ãons corresponderam aos resultados obtidos no estudo de metal simples, fixando o valor de pH 2,0 para precipitaÃÃo do Cu2+, pH entre 4,0 e 5,0 para precipitaÃÃo do Zn2+ e pH = 6,0 para precipitaÃÃo do Ni2+. Para os sistemas Cu2+-Zn2+ e Cu2+-Ni2+ os resultados mostraram pouca interferÃncia de um Ãon na precipitaÃÃo do outro, sendo possÃvel a separaÃÃo de ambos e precipitaÃÃo acima de 90%. Por outro lado para o estudo do sistema Zn+2-Ni +2 foi constatado que o controle rÃgido de pH à determinante para separar sulfetos metÃlicos com pKps muito prÃximos, obtendo 90% de remoÃÃo para ambos. Os resultados envolvendo a separaÃÃo fracionada do sistema Cu2+-Zn2+-Ni2+ foram satisfatÃrios, nÃo necessitando do uso de agentes complexantes e sim o controle rÃgido de pH. / The precipitation of metal sulfides is an important process to treat wastewater coming from industrial effluents from metal finishing Mostly because it is a viable alternative for the recovery and reuse of metals by controlling the pH becomes selective. This work studied the selective precipitation.of Cu2+, Zn2+-and Ni2+ using H2S(g) provided by the hydrolysis of thioacetamide. The studies were conducted in laboratory scale and in two steps. Firstly, studies were performed for each single metal ion (100 mg.L-1) were initially investigated the following parameters: setting up a reaction system, the pH range (0,5 a 6,0) and exposure time (30 -150 minutes). The studies showed that Cu2+ easily precipitated in the first 30 min at all pH values tested reaching 99,9% of removal. For the Zn2+ precipitation is preferred when the pH was between4.0 and 5.0, and optimum exposure time was 60 min, so that the precipitation was greater than 95.0%. The pH has influenced the process ion precipitation, and most influential for the Ni2+, starting from their precipitation at pH 6.0, and after 4 times of 30 min, adding an exposure time of 120 min so that its removal should be above 90%. In the second step, studies were conducted in binary and ternary mixture of ions from the values of pH and exposure time for individual study found. In summary, the results obtained in experiments conducted with the mixture of the ions correspond to the results obtained in the study of single metal, setting the pH value of 2.0 to precipitation of Cu2+, pH between 4.0 and 5.0 for precipitation of Zn2+ and pH = 6,0 for precipitation of Ni2+. For systems Cu2+ and Zn2+, Cu2+ and Ni2+ results showed little interference of an ion in the precipitation of another, with the possible separation and precipitation of both above 90%. In addition to the study of the system Zn2+- Ni2+ was found that the rigid control of pH is critical to separate metallic sulfides pKps very close to getting 90% removal for both. The results involving the fractional separation system Cu2+, Zn2+ e Ni2+ were satisfactory and does not require the use of complexing agents, but the drive control of pH.

No description available.