Plasmid DNA/RNA separation by ultrafiltration: modeling and application study

RNA is one of the main soluble contaminants that needs to be separated from plasmid DNA (pDNA) during its recovery process from fermentation broths. Although significantly different in molecular size, pDNA and RNA are difficult to separate by membrane filtration due to the fact that both types of molecules are highly deformable and therefore suffer flow-induced elongation when permeating through porous membranes. The possibility of performing this separation by ultrafiltration is investigated here with the aid of a theoretical model describing the interactions between two electrically charged, flexible macromolecules that simultaneously permeate through a porous membrane. The results of the simulations, applied to pDNA and the different types of RNA present in cell lysates under typical process conditions, show that only by a careful choice of the membrane pore size and the imposed permeate flux one can achieve the required selectivity in this operation. Ultrafiltration tests using microfiltered lysates from the production of two different plasmids, pVAX1-LacZ (6050 bp) and pCAMBIA-1303 (12,361 bp), were carried out to check the validity of the theoretical predictions; the experimental results confirm these predictions and the idea that this technique can be used in practice for pDNA purification.Fundação para a Ciência e a Tecnologia (FCT

A bi-layer electrospun nanofiltration membrane for plasmid DNA recovery from fermentation broths

The 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.This work was supported by the Portuguese Foundation for Science and Technology (FCT), (PTDC/EME-TME/103375/2008 and PTDC/EBB-BIO/114320/2009). To Ricardo Fradique for helping in the production of the graphical abstract