Design of expanded bed supports for the recovery of plasmid DNA by anion exchange adsorption.
In this study we detail the rational design of new chromatographic adsorbents tailored for the capture of plasmid DNA. Features present on current chromatographic supports that can significantly enhance plasmid binding capacity have been identified in packed bed chromatography experiments and blueprints for improved expanded bed adsorbents have been put forward. The characterisation and testing of small (20-40 microm) high density (>3.7 g cm(-3)) pellicular expanded bed materials functionalised with various anion exchange structures is presented. In studies with calf thymus DNA, dynamic binding capacities of 1.2 and 3.4 mg ml(-1) were recorded for prototype diethylaminoethyl-and polyethylene imine-linked adsorbents which were respectively 25 and 70 fold higher than those of equivalently derivatised commercial expanded bed materials. The prototype polyethylene imine-coupled material exhibited severe sensitivity to inter-particle bridging by nucleic acid polymers, gave low DNA recoveries (<37%) and proved difficult to regenerate. In contrast, few operational difficulties were experienced with the diethylaminoethyl-linked prototype adsorbent and successful high capacity (>0.8 mg ml(-1)) capture of plasmid DNA from crude neutralised E. coli lysate was demonstrated.
Theodossiou
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I
Center for Process Biotechnology, BioCentrum-DTU, Technical University of Denmark, Lyngby.
Søndergaard
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M
Thomas
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OR
D000327:Adsorption; D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D010957:Plasmids
D004247:DNA