Continuous isolation of plasmid DNA by annular chromatography

Abstract

Continuous chromatographic separations, especially of multicomponent mixtures, constitute interesting options for biotechnological downstream processing. Taking the separation of plasmid DNA from clearified lysates on hydroxyapatite as a pertinent example, we discuss the potential of continuous annular chromatography (CAC) in comparison with conventional (preparative) batch chromatography. In CAC the column is realized in the form of a thin (5 mm, height 210 mm) slowly rotating annulus. The performance of such a CAC column is compared to that of an ("analytical") batch column of similar thickness (diameter) and length (4 x 250 mm) and that of a ("preparative") batch column of similar cross-sectional surface area and height (50 x 210 mm). The quality of the obtained plasmid as defined by the appearance of the corresponding agarose gels (native and linearized plasmid), the 260/280 ratio and the biological activity (transient transfection of HEK 293 cells) was found to be identical in all three cases. The yields are also shown to be equivalent. The loading factor is found to be the most decisive parameter for the transfer of a given separation method between the continuous and the batch columns. Under nonoptimized conditions, plate numbers tended to be lower in the continuous compared to the batch columns. This is shown to be largely due to an artifact created by the CAC design (collection of averaged fractions at the outlets) and can be overcome by optimizing the rotation speed. Surprisingly the large batch column consistently gave better plate numbers than either the small batch or the CAC column. Compared to the preparative batch column, wall effects are more pronounced in the CAC (respectively the small diameter batch column), which may translate into better bed stability but conceivably also contributes to an increase in plate height, due to the reduction in bed density usually observed in the proximity of the wall. The CAC is shown to be a powerful approach to continuous chromatography, which allows a direct and straightforward upscale of chromatographic bioseparation methods.

Continuous Matrix‐assisted Refolding of Inclusion‐body Proteins: Effect of Recycling

“…The dimensionless column loading factor, LF, a common parameter in conventional batch chromatography, which is used whenever columns of different sizing are to be compared, was found to be most useful for maintaining the same efficiency during method transfer from discontinuous to continuous operation [18]. The dimensionless loading factor LF for the PCAC system can be expressed as the ratio of sample volume to the column volume (CV) and is given by LF F´21600 Vx (16) where F is the feed flow rate applied onto the rotating bed and V is the volume of the stationary phase.…”

Adsorption of plasmid DNA on ceramic hydroxyapatite chromatographic materials

“…Many applications of hydroxyapatite were described for protein and DNA purification [6,[11][12][13][14]. Hydroxyapatite synthesized by the Tiselius method resulted in formation of unstable rectangular plate-shaped crystals with poor flow properties and high-pressure drop [15].…”

Performance of R‐N(R′)‐R′′ functionalised poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) monolithic sorbent for plasmid DNA adsorption

“…Chromatographic techniques usually employed include: size-exclusion [9][10][11], RP [12,13], hydrophobic interaction [14][15][16], hydroxyapatite [17][18][19] and triple-helix affinity [20,21]. Anion-exchange chromatography is the most versatile technique as it offers the advantages of rapid separation, no solvent requirement, sanitisation with NaOH and a wide selection of industrial media.…”