Separation of plasmid DNA from protein and bacterial lipopolysaccharides using displacement chromatography

The preparation of plasmid DNA at large scale constitutes a pressing proble m in bioseparation. This paper describes a first investigation of displacem ent chromatography as a means to separate plasmid DNA (4.7 kb) from E. coli lipopolysaccharides and protein (holo transferrin), respectively. Displace ment chromatography has advantages in this regard, since the substance mixt ure is resolved into rectangular zones of the individual components rather than into peaks. Thus a higher total concentration can be maintained in the pooled product fractions. Hydroxyapatite (type I and II) and anion exchang e stationary phases were included in the experiments. In addition to a conv entional anion exchange column packed with porous particles, the recently i ntroduced continuous bed UNO TM anion exchange column was investigated. No DNA purification was possible with either hydroxyapatite material. Conventi onal particle based columns in general were not suited to the separation of any two substances varying considerably in molecular mass, e.g. plasmid DN A and standard protein. Presumably, the direct competition for the binding sites, which is essential in displacement chromatography, was restricted by the size dependency of the accessible stationary phase surface area in thi s case. Better results were obtained with the continuous bed column, in whi ch the adsorptive surface coincides with the walls of the flow through pore s. As a result the accessible surface does not vary as much with the size o f the interacting molecules as for the conventional stationary phase materi als. Sharper transitions were also observed between substance zones recover ed from the UNO TM column. The steric mass action model was used to aid met hod development in case of the anion exchange approach. While further resea rch in obviously necessary, displacement chromatography on continuous bed c olumns has been shown to be capable of separating plasmid DNA from typical impurities.