Plasmid DNA recovery from fermentation broths by a combined process of micro- and ultrafiltration: Modeling and application
2012
Nunes, José C. | Morão, António M. | Nunes, Catherine | Pessoa de Amorim, Maria Teresa | Escobar, Isabel C. | Queiroz, João A.
Microfiltration and ultrafiltration operations were used in tandem to isolate and purify a 6050 bp plasmid DNA (pDNA). To achieve primary isolation of the plasmid from fermentation broths, immediately after cell lysis, a 0.2 μm microfiltration membrane was selected for solid/liquid separation, which was performed in a diafiltration mode, as an alternative to centrifugation. Then to attain plasmid concentration and purification, an ultrafiltration membrane with a pore radius of 4.1 nm was selected. Permeation of pDNA and RNA in the two membrane steps was modeled using recently published mass transfer models applicable to the permeation of closed segmented chains and freely-jointed chains, respectively. The permeation of proteins and genomic DNA (gDNA) was also studied in these operations. The microfiltration operation allowed high plasmid and RNA permeation, as expected. It was observed that significant amounts of gDNA, previously precipitated during the cell lysis step, reappear in solution during the diafiltration step. The effect of the ionic strength on this apparent re-solubilization was investigated, by testing the addition of two different salts to the diafiltration buffer: CH₃COOK and CaCl₂. The results show that these salts can be used to control gDNA apparent re-solubilization. During the ultrafiltration operation high plasmid retention with low adsorption was obtained under low ionic strength conditions. The results also show that a significant removal proteins and the re-solubilized gDNA is achieved, as well as some purification in respect to low molecular weight RNA, since all these components permeate through the ultrafiltration membrane.
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