Browsing by Author "Castilho, Pedro Henrique Barata"
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- A bi-layer electrospun nanofiber membrane for plasmid DNA recovery from fermentation brothsPublication . Correia, Tiago R.; Antunes, Bernardo Paiva; Castilho, Pedro Henrique Barata; Nunes, José; Amorim, Maria T. Pessoa de; Escobar, Isabel; Queiroz, João; Correia, I.J.; Morão, AntónioThe demanding ever-increasing quantities of highly purified biomolecules by bio-industries, has triggered the development of new, more efficient, purification techniques. The application of membrane-based technologies has become very attractive in this field, for their high throughput capability, simplicity of operation and scale-up. Herein we report the production of a bi-layer membrane by electrospinning (ES), in which a support of poly ε-caprolactone nanofibers was coated with a polyethylene oxide/sodium alginate layer, and subsequently cross-linked with calcium chloride. The membranes were characterized by SEM, ATR-FTIR, contact angle measurements, and were applied in the recovery process of a plasmid. The results show that membranes retained the suspended solids while allowing the permeation of plasmid DNA, with high recovery yields and improved RNA retention. Moreover, they also showed a very low fouling tendency. To the best of our knowledge it is the first time that ES membranes are applied in this type of bioprocess.
- Modification of microfiltration membranes by hydrogel impregnation for pDNA purificationPublication . Castilho, Pedro Henrique Barata; Correia, Tiago R.; Amorim, Maria T. Pessoa de; Escobar, Isabel; Queiroz, João; Correia, Ilídio Joaquim Sobreira; Morão, AntónioThe huge efforts for the achievement of highly purified biomolecules are growing every day. A great number of efficient techniques, such as chromatography, are already available in laboratory for separation processes. However, membrane‐based technologies are the best match to assure simplicity, efficiency and easy scale‐up procedures. Herein we report the modification of a commercial microfiltration membrane for plasmid DNA purification by agarose gel impregnation. The membrane was characterized by SEM, ATR‐FTIR, EDS, contact angle, and porosity measurements. Additionally, the membrane pore radius was estimated from observed rejections of different proteins and with that information the rejection of a 6050 bp plasmid DNA (pDNA) molecule was estimated for different values of flux using a theoretical model of large flexible molecules in membranes with parallel cylindrical pores, which is applicable to pDNA ultrafiltration in conventional membranes, as recently shown in the literature. The experimental results show that the modified membrane has higher pDNA rejections than the predicted by the model, suggesting that the different type of porous structure that a hydrogel has, may have a positive effect on pDNA rejections as compared to other biomolecules with more rigid structures, making this type of modified membranes potential better candidates to be used for the selective recovery of pDNA in this type of bioprocesses.
- Production of membranes for filtration of biomoleculesPublication . Castilho, Pedro Henrique Barata; Morão, António Miguel Parreira Cabral Forjaz; Correia, Ilídio Joaquim SobreiraPlasmids are autonomously replicating entities that can be found in all bacterial species and contribute for bacterial adaption and evolution. The demand of highly purified biomolecules has triggered the development of new separation technologies. Herein, plasmid DNA (pDNA) purification process has been extensively investigated, in order to obtain highly purified molecules for gene therapy applications and DNA (deoxyribonucleic acid) vaccines. The purification of plasmid DNA is currently performed by different techniques, namely chromatography (anion-exchange, hydrophobic interaction, reversed phase, affinity and sizeexclusion), enzymatic and membrane processes. Membrane technology is a broad and highly interdisciplinary field, where process engineering, material science and chemistry meet to produce membranes that have a wide range of applications, such as water, biomolecules and plasmid DNA purification. Furthermore, membrane systems take advantage of their selectivity, high surface-area-per-unit-volume. Herein, the main goal was to produce membrane systems - electrospinning membranes: poly ε-caprolactone, polyethylene oxide and k-carrageenan; modified-nylon membranes: nylon membrane impregnated with agarose - in order to perform microfiltration and ultrafiltration processes, respectively. The produced membranes were characterized by Scanning Electron Microscopy, Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy and Energy Dispersive Spectroscopy. The water contact angles were also determined and the results obtained showed that the produced membranes presented a hydrophilic character, which is in agreement with the date previously described in literature. Porosity studies were also performed and the results demonstrated that the electrospun membranes have porosity around to 80% and the modified-nylon membranes have porosities of approximately 40%. These values can be considered to be very high, when comparing these membranes to conventional microfiltration and ultrafiltration membranes produced by phase inversion. The plasmid DNA rejection was determined on the membranes produced and the experimental results showed that the modified-nylon membrane presented rejection values up to 100%, depending on the imposed permeate flux, which is an attractive feature for its application on plasmid DNA purification by ultrafiltration. In respect to the electrospun membranes produced the observed rejections were found to be lower, up to 30%, which demonstrates that this membranes need to be optimized or modified (post-synthesis modification).