Adsorption of plasmid DNA on ceramic hydroxyapatite chromatographic materials.

The adsorption of plasmid DNA onto two different types of ceramic hydroxyapatite beads with a particle diameter of 20 µm, namely Ceramic Hydroxyapatite Type II and the Type III, which is not commercially available, were investigated. Type II and the Type III have a pore diameter of 80 and 240 nm, respectively. Equilibrium and dynamic binding capacity for a 4.9 kbp model plasmid on Ceramic Hydroxyapatite Type II and Type III were enhanced by addition of NaCl to the adsorption buffer. This result indicates that the adsorption mechanism cannot be solely explained by electrostatic interaction. The affinities of plasmid DNA for Ceramic Hydroxyapatite Type II (with a K(D) of ≈0.005 mg/mL) and to Hydroxyapatite Type III (with a K(D) of ≈0.045 mg/mL) were not affected by NaCl, whereas the binding capacity was. This observation corroborates the assumption that a change of the shape of the plasmid molecule is affected and could be the reason for increased binding capacity with salt. The maximal binding capacity shows that at least a part of the CHT II bead must be accessible for the plasmid, whereas CHT III can be saturated with the plasmid. In both cases, an extremely hindered transport takes place.

Adsorption of pDNA on microparticulate charged surface.

Plasmid DNA purification strategies are often based on chromatographic processes, which often suffer from low dynamic binding capacities and poor productivity. The limitations of conventional chromatography media for adsorption of pDNA led us to the investigation of irregular polymeric microparticles with quaternary amine functionality. They have an average particle size of 8-10 microm. The adsorption properties of microparticles are comparable to monolithic supports, i.e. the adsorption process is accomplished within 15 min and the binding capacity is in the range of 15 mg/mL. Desorption with NaCl renders nearly theoretical yield. Due to the small particle size the mode of operation is rather batch adsorption than column chromatography. The phase separation is facilitated by formation of reversible flocs of pDNA and microparticles. The implementation of microparticles in a lab-scale capture step out of processed E. coli lysates demonstrated their potential as a high speed and high capacity material for pDNA purification.