Ultrafiltration of supercoiled plasmid DNA: Modeling and application

Abstract

A mass transfer model is proposed for predicting sieving coefficients, S obs , of supercoiled plasmid DNA (pDNA), in the presence of a salt, in membranes with narrow pores, i.e., pores smaller than the gyration radii of the plasmids to be considered for a certain application. The model assumes that permeation occurs due to plasmid suction at the membrane surface as a result of the convective flow, being the probability of permeation also dependent on the instantaneous molecular conformation of the plasmid, when getting close to the pore. Two different approaches are tested to model plasmid structure, that of a closed segmented chains (CSC) of double stranded DNA, and that of considering the superhelical chain as a freely jointed chains (FJC). Both approaches were used to estimate the radius of gyration, r g , of different plasmids by statistical simulation, and the obtained values were compared with experimental data available in the literature. A 6050 bp plasmid, pVAX1-LacZ, was used in the experimental work, in which filtration tests were performed using three different ultrafiltration membranes of known pore size, in a 10 ml stirred cell. At constant ionic strength, sieving coefficients were determined as a function of the permeate flux, J v , at two different values of stirring speed, ω. The results are in very good agreement with the model predictions at the highest stirring speed and the observed deviations found at the lowest stirring speed were interpreted with the aid of the developed model by considering the possibility of plasmid adsorption. Then, it was investigated the effect of changing the ionic strength of the medium at constant J v and ω. The obtained results clearly agree with the model predictions.

Modification of microfiltration membranes by hydrogel impregnation for pDNA purification

“…As can be seen, at low fluxes the modified‐Nylaflo membrane presented near 100% of pDNA rejection and the rejection decreases as the flux is increased, despite the fact that the hydrodynamic radius of the plasmid used in these tests, pVAX1‐LacZ, is near 83 nm (value estimated from the diffusion coefficient of the plasmid using the Stokes–Einstein equation), i.e., much higher than the pore radius of the membrane. This type of behavior has been previously reported in literature for pDNA and can be interpreted as a result of flow‐induced deformation of the molecular structure of these macromolecules which leads to their permeation through membranes with narrow pores.…”

“…Knowing the r p value of the membrane and the radius of gyration, r g , of the pDNA molecule, Morão et al have recently shown that one can accurately estimate observed rejections of this type of flexible molecules in the case of several conventional (asymmetric polymeric) ultrafiltration membranes . This model was originally established for linear molecules, namely linear dextran and linear double‐stranded DNA and then adapted to supercoiled plasmid DNA.…”

Advances in chromatographic supports for pharmaceutical‐grade plasmid DNA purification

“…Membrane technology has been widely investigated and improved in recent years, becoming established either for ultrafiltration or purification steps in downstream processing of pDNA. Morao et al recently proposed a mass transfer model for predicting sieving coefficients of sc pDNA by ultrafiltration with narrow pore membranes . Indeed, membrane chromatography has also gained impact in the recovery of pDNA from a complex E. coli lysate.…”

Supercoiled plasmid DNA purification by integrating membrane technology with a monolithic chromatography

“…The application of membrane systems for pDNA purification has been investigated and used in biological and chemical industries . Microfiltration is a membrane process that is commonly employed in several applications such as solid–liquid separation .…”