Adsorption of plasmid DNA on ceramic hydroxyapatite chromatographic materials

Abstract

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.

Dissociation of Antibody–Contaminant Complexes With Hydroxyapatite

“…At low conductivity, mutual electrostatic repulsion between HA phosphates and DNA phosphates is believed to limit the ability of DNA to bind to HA calcium. [17,18] That repulsion is suppressed at high conductivity, allowing DNA to interact more freely with apatite calcium. In addition, mutual repulsion among DNA phosphates is reduced, making DNA more flexible and better able to conform to the HA surface.…”

Next generation vaccines and vectors: Designing downstream processes for recombinant protein‐based virus‐like particles

“…Industrial intermediate purification of e.g., human papilloma‐VLPs has been performed with CHT chromatography subsequent to a CEC capture step [91, 121]. CHT chromatography is a mixed‐mode chromatography method often used for large biomolecules with typical pore sizes of 80 to 100 nm [122].…”

Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography

“…In this context, Giovannini and Freitag [75] were among the first to investigate HA for pDNA purification as a function of geometrical properties, using HA materials prepared at different sintering temperatures.The accessibility of the surface (pore size) and also the P/C-site ratio were discussed as capacity-determining factors. Jungbauer et al [76] recently compared a novel-potentially more DNA compatible-HA material with an average pore size of 240 nm (particle diameter of 20 µm) with standard Bio-Rad type II material (pore size 80 nm, particle diameter 20 µm). Capacities increased considerably for the novel material; however, the chromatographic process was still characterised by extremely hindered mass transport.…”