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.

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.

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.

No description available.

M. Tech. (Department of Chemical Engineering, Faculty of Engineering and Technology), Vaal University of Technology. / Heavy metal ions are one of the most toxic materials in the environment. Adsorption is the most used process for the removal of heavy metals from wastewater. Much research has been conducted into processes to remove heavy metals using different adsorbents. Various adsorbents have been used to remove heavy metal ions from wastewater especially those that are harmful to mankind. Zeolite, clay, activated carbon and biopolymers are the most common adsorbents used. In this research, gelatin, and cellulose nanocrystals (CNCs) were used to synthesize a hydrogel membrane to remove Cu(II) and Co(II) metal ions from mining processes wastewater. The synthetic wastewater was prepared in the laboratory to conduct the experiments. Batch experiments were conducted to obtain the optimum conditions for the Cu(II) and Co(II) metal ions. The effect of parameters such as pH, ratio, contact time, and temperature were also determined. The optimum conditions obtained were 120 min contact time for both metal ions at the temperature of 30oC, pH 5 for copper and pH 7 for cobalt. The high removal of both metals ions was obtained using the ratio 3:1 (75% Gelatin and 25% CNCs) at the temperature of 303K. The maximum adsorption capacity of Cu(II) and Co(II) was 7.6923 mg/g and 10.988 mg/g, respectively. The high percentage removal of Cu(II) and Co(II) metal ions obtained was found to be 70.5% for Cu(II) at pH 5 and 74.5% for Co(II) at pH 7. The experimental data fit well to Pseudo-first-order kinetic and Freundlich isotherm models (KF= 1.89x103 mg/g for copper and 3.7x102 mg/g for cobalt) for both metal ions. The values of energy (E) from D-R model have shown that the adsorption of both metal ions was of physical nature (E<8kJ/mol) then confirmed by the thermodynamic results (ΔH°). The kinetic diffusion models have shown that the experimental data fit well with the film diffusion (R2= 0.977 and 0.989) for both metal ions at pH 5. Negative values of ΔG°obtained for both metal ions indicate that the adsorption process was spontaneous. The positive values of ΔH° obtained showed a physical adsorption process and also indicate that the adsorption process of both metal ions was endothermic. The positive values of ΔS° indicate an increase in randomness at the solid/solution interface during adsorption.