Plasmid purification using non-porous anion-exchange silica fibres

• A new type of fibre-based anion-exchange material for plasmid purification was developed. The basic material consisted of non-porous silica fibres with a mean diameter of 1.5 mu m and a surface area of 2.4 m(2) g(-1). The fibre surface was provided with several types of ligands, either by adsorption of polymers (chitosan or poly(ethyleneinune)) or by polymerization of amine-containing acrylic monomers onto a propyl methacrylatesilanized surface. The resulting polymer layers contained primary, tertiary or quaternary amines as ion-exchange groups. The packing density could be varied considerably, 9-34% (v/v). The loose packing structure provided excellent flow properties suitable for high-speed operations. The best overall performance was shown by silica fibres provided with tertiary amine polymers, having a plasmid-binding capacity of 0.9 mg ml(-1) (pre-purified plasmid) and a plasmid recovery of 62% (performance data remained stable though several adsorption cycles). The high flow rates possible with the fibre material made it especially useful when large volumes of cleared lysate were processed. The columns could be operated with retention of their adsorption properties at speeds of up to 1800 cm h(-1) equivalent to 0.5 column volumes per minute. The binding capacity was found to be lower than anticipated from the design of the fibres. Fluorescence imaging showing individual plasmid molecules indicated the fibre population to be heterogeneous with respect to plasmid adsorption, some fibres displaying poor binding properties. Possible reasons for this heterogeneity are discussed. (c) 2007 Elsevier B.V. All rights reserved.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

plasmid

silica fibres

anion-exchange chromatography

plasmid DNA

plasmid purification

imaging

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