Purification of plasmid DNA from Escherichia coli ferments using anion-exchange membrane and hydrophobic chromatography.

A novel downstream bioprocess was developed to obtain purified plasmid DNA (pDNA) from Escherichia coli ferments. The intermediate recovery and purification of the pDNA in cell lysate was conducted using hollow-fiber tangential filtration and frontal anion-exchange membrane and elution hydrophobic chromatographies. The purity of the solutions of pDNA obtained during each process stage was investigated. The results show that the pDNA solution purity increased 30-fold and more than 99% of RNA in the lysate was removed during the process operations. The combination of membrane operations and hydrophobic interaction chromatography resulted in an efficient way to recover pDNA from cell lysates. A better understanding of membrane-based technology for the purification of pDNA from clarified E. coli lysate was developed in this research.

Purification of plasmid DNA using tangential flow filtration and tandem anion-exchange membrane chromatography.

A new bioprocess using mainly membrane operations to obtain purified plasmid DNA from Escherechia coli ferments was developed. The intermediate recovery and purification of the plasmid DNA in cell lysate was conducted using hollow-fiber tangential filtration and tandem anion-exchange membrane chromatography. The purity of the solutions of plasmid DNA obtained during each process stage was investigated. The results show that more than 97% of RNA in the lysate was removed during the process operations and that the plasmid DNA solution purity increased 28-fold. One of the main characteristics of the developed process is to avoid the use of large quantities of precipitating agents such as salts or alcohols. A better understanding of membrane-based technology for the purification of plasmid DNA from clarified E. coli lysate was developed in this research. The convenience of anion-exchange membranes, configured in ready-to-use devices can further simplify large-scale plasmid purification strategies.

Breakthrough performance of plasmid DNA on ion-exchange membrane columns.

Breakthrough performance of plasmid DNA adsorption on ion-exchange membrane columns was theoretically and experimentally investigated using batch and fixed-bed systems. System dispersion curves showed the absence of flow non-idealities in the experimental arrangement. Breakthrough curves (BTC) were significantly affected by inlet flow rate and solute concentration. In the theoretical analysis, a model was integrated by the serial coupling of the membrane transport model and the system dispersion model. A transport model that considers finite kinetic rate and column dispersed flow was used in the study. A simplex optimization routine, coupled to the solution of the partial differential model equations, was employed to estimate the maximum adsorption capacity constant, the equilibrium desorption constant, and the forward interaction rate constant, which are the parameters of the membrane transport model. The analysis shows that as inlet concentration or flow rate increases, the deviation of the model from the experimental behavior decreases. The BTCs displacement as inlet concentration increases was explained in terms of a greater degree of column saturation reached and more efficient operation accomplished. The degree of column saturation was not influenced by inlet flow rate. It was necessary to consider in the column model the slight variation in the BTC produced by the axial dispersion, in order to accomplish the experimental curve dispersion. Consequently, the design criteria that for Pe > 40 the column axial dispersion can be neglected should be taken with precaution.