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The possible use of membrane filtration in the recovery process of plasmid DNA (pDNA) from fermentation broths, for applications in gene therapy and DNA vaccine applications, has been studied in recent years. Application has been made to achieve efficient clarification from suspended solids after cell lysis and also to remove hydrophobic soluble contaminants such as RNA [1-5]. In our recent research, microfiltration and ultrafiltration have been performed in tandem to pre-purify and concentrate the plasmid before chromatography [6].
The modification of a microfiltration membrane by deposition of a film of electrospun polycaprolactone (PCL) nanofibers is being studied as a method for obtaining ultrafiltration membranes with adequate pore size, to retain the pDNA molecules. Polymer nanofibers are widely known for their potential applications in the fields of catalysis, electronics, protective clothing, nanocomposites, filtration, bioengineering and biotechnology [7]. Electrospinning is one of the techniques that has been widely used for the fabrication of nanofibers from various synthetic and natural polymers.
In a typical electrospinning process the deposited fiber mesh obtained is generally highly porous and with an open structure [8]; therefore, it can be expected that less molecular deformation of pDNA molecules occurs, when comparing these membranes to conventional asymmetric porous membranes prepared by phase inversion. This can be a very important characteristic of these films for their application for pDNA concentration and simultaneous purification, provided that very thin fibers can be formed. The ability of obtaining nanofibers as thin as 100 nm found in preliminary tests carried out with PCL enabled a considerable increase in pDNA retention, in respect to a unmodified microfiltration membrane, FSM0.45PP from DSS/Alfa Laval. The possibility of further modifying this polymer by a suitable chemical method, in future work, motivates our present research.
References:
[1] D.W. Kahn, M.D. Butler, D.L. Cohen, M. Gordon, J.W. Kahn, M.E. Winkler, Purification of plasmid DNA by tangential flow filtration, Biotechnol Bioeng, 69 (2000) 101-106.
[2] D. Kendall, G.J. Lye, M.S. Levy, Purification of plasmid DNA by an integrated operation comprising tangential flow filtration and nitrocellulose adsorption, Biotechnology and Bioenginering, 79 (2002) 816-822.
[3] A. Eon-Duval, R.H. MacDuff, C.A. Fisher, M.J. Harris, C. Brook, Removal of RNA impurities by tangential flow filtration in an RNase-free plasmid DNA purification process, Anal Biochem, 316 (2003) 66-73.
[4] P. Guerrero-Germán, R. M. Montesinos-Cisneros, D.M.F. Prazeres, A. Tejeda-Mansir, Purification of plasmid DNA from Escherichia coli ferments using anion-exchange membrane and hydrophobic chromatography, Biotechnology and Applied Biochemistry, 58 (2011) 68–74.
[5] I. Theodossiou, I.J. Collins, J.M. Ward, O.R.T. Thomas, P. Dunnill, The processing of a plasmid-based gene from E-coli. Primary recovery by filtration, Bioprocess Eng, 16 (1997) 175-183.
[6] J. C. Nunes, A. Morão, Catherine Nunes, M. T. Pessoa de Amorim, I. C. Escobar, J. A. Queiroz, Plasmid DNA recovery from fermentation broths by a combined process of micro- and ultrafiltration: modeling and application, submitted to Journal of Membrane Science (2012), under review.
[7] S. Kaur, D. Rana, T. Matsuura, S. Sundarrajan, S. Ramakrishna, Preparation and Characterization of Surface Modified Electrospun Membranes for Higher Filtration Flux, Journal of Membrane Science, in Press, doi:10.1016/j.memsci.2011.11.045 (2011).
[8] Z. Zhao, J. Zheng, M. Wang, H. Zhang, C.C. Han, High performance ultrafiltration membrane based on modified chitosan coating and electrospun nanofibrous PVDF scaffolds, Journal of Membrane Science, 394 (2012) 209– 217.