Thiophilic interaction chromatography for supercoiled plasmid DNA purification

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Abstract

Supercoiled plasmid DNA was selectively purified from its open circular form by thiophilic interaction chromatography, performed in the presence of high concentrations of water-structuring salts. To identify optimal conditions for purification, various aromatic thioether ligands were coupled to a chromatographic support and screened for their ability to separate plasmid isoforms from each other and from other host cell contaminants, including RNA, genomic DNA, protein, and endotoxins. Selectivity of the chromatographic medium depended on the structure of the ligands, with characteristics of the substituents on the aromatic ring determining the resolution between the different plasmid DNA isoforms. Optimal resolution was obtained with ligands consisting of an thioaromate, substituted with highly electronegative groups. When 2-mercaptopyridine was used as a ligand, the difference in conductivity for eluting open circular and supercoiled plasmid DNA is only 6 mS/cm. However, with 4-nitrothiophol the resolution for plasmid DNA separation on the media increased, resulting in a 20 mS/cm difference. When used in combination with a prior group separation step, these aromatic thioether ligands facilitated the isolation of highly purified supercoiled plasmid DNA, suitable for use in gene therapy and DNA vaccine applications.

Introduction

The demand for production of plasmid DNA has increased greatly in response to rapid advances in the use of gene therapy and plasmid DNA vaccines. By introducing genetic material into target cells of the body, gene therapy aims to restore, cancel, or enhance gene function, and ultimately alter a physiological function (Cannon and Anderson, 2000). In current gene therapy trials, vehicles introducing the genetic material into the body often are viral vectors. However, recent studies on the possible use of retrovirus (Schroder et al., 2002) and adeno-associated virus (Nakai et al., 2003) for DNA transport show a clear danger for integration of the vector in coding regions of the host genome. The recent drawbacks in the use of retroviral vectors for treatment of SCID resulted in a temporary hold by the FDA on all ongoing clinical trials with retrovirus (Check, 2002), while various gene-delivery systems can be used, non-viral vectors, in the form of plasmid DNA, are often preferred in clinical vaccine applications to minimize the risk of viral infections (Jolly, 1994). Vaccination with plasmid DNA shows little risk for reversion to a disease-causing form, and results in both humoral and cellular immune responses. Furthermore, plasmid DNA is more stable and easier to store compared to traditional viral vaccines (Robinson, 1999; Gurunathan et al., 2000a, Gurunathan et al., 2000b; Kim and Weiner, 2000; Riemann et al., 2001).

The efficacy of plasmid DNA vaccination depends on the endogenous production of an epitope, resulting in a strong host B-cell response and ultimately, production of antibodies. By combining different genes, poly-epitope vaccines can be produced. A major advantage of such plasmid-based vaccines lies in the cost-effective and generic production of plasmid DNA. However, to satisfy strict health guidelines, this material must be available in large amounts as highly purified, homogeneous preparations of supercoiled plasmid DNA (Center for Biologics Evaluation and Research, CBER, 1996, CBER, 1998; Committee for Proprietary Medicinal Products, CPMP, 2001). Therefore, careful analysis of the plasmid DNA topology is required. Several methods are currently available and although agarose gel electrophoresis here probably still is the standard because of its low costs, capillary gel electrophoresis might be the preferred method here because of its high selectivity and sensitivity (Schmidt et al., 2001).

Plasmids are circular, double-stranded DNA molecules that comprise approximately 1% of the total content of the host bacterial cell (Table 1). They are generally isolated by an alkaline lysis procedure, designed to disrupt the host cells and denature proteins and genomic DNA, while preserving plasmid structural integrity (Diogo et al., 1999). While the majority of plasmids isolated from bacterial cells are covalently closed circles (ccc) which are negatively supercoiled, breakage of one of the DNA strands during growth and lysis or during purification results in loss of molecular coiling and conversion of the plasmid to an open circular or relaxed form (Schmidt et al., 2001). Because of the similarity in chemical composition and structure with plasmid DNA, and its high abundance in crude plasmid preparations, RNA removal presents a true challenge in the production of genetic therapeutics (Atkinson and Mavituna, 1991). RNA can be removed by several techniques, including treatment with RNase A. Because of the inherent risk of using bovine-derived products means, such products are not recommended for pharmaceutical production intended for humans. Therefore, other sources for RNase are now under investigation (Cooke et al., 2001). Other techniques used in large-scale processes include selective precipitation, filtration, aqueous two-phase extraction and several chromatographic techniques such as size-exclusion, reversed phase, hydrophobic interaction, hydroxyapatite, anion exchange, silica and triple-helix chromatography (Eon-Duval, 2003). A recent study describes an interesting non-enzymatic approach to RNA removal that is based on size exclusion chromatography (Lemmens et al., 2003). Using this approach, a sample containing plasmid DNA can be conditioned in one step for the subsequent chromatographic purification step.

It is generally believed that the supercoiled form of plasmid DNA is the physiologically active conformation, and it is this form that is optimal for cell transfection (Kano et al., 1981, Sekiguchi and Kmiec, 1989). Furthermore, regulatory authorities require that plasmid DNA solutions destined for therapeutic use contain reproducible amounts of the open circular and supercoiled forms. Because it is difficult to control shearing stress during cell lysis and upstream processing, it is preferable to remove any resulting open circular plasmid DNA in a downstream purification step, such that a homogeneous supercoiled plasmid DNA solution is produced. Consequently, plasmid purification protocols must be robust and amenable to scale-up for production purposes, and must reproducibly and selectively purify the supercoiled form of the plasmid. Although a variety of methods are available, few are useful for preparative-scale purification, and few yield preparations that are homogeneous for the supercoiled form of plasmid DNA. In previous studies (Lemmens et al., 2003), we showed that aromatic thioether-containing chromatographic adsorbents can be used to separate the supercoiled and open circular forms of plasmid DNA in the presence of high concentrations of lyotropic or water-structuring salts, such as ammonium sulfate. While the mechanism underlying the salts’ effect is not clear, it is possible that they may differentially compact the various nucleic acid moieties, inducing conformational changes that can be exploited for purification. We found that for selective retention of supercoiled plasmid DNA, the chromatographic medium had to contain, at a minimum, an aromatic ring structure and a thioether moiety. In this work, we have screened additional aromatic thioether adsorbents to identify ligands with even greater plasmid DNA isoform separation properties.

Section snippets

Materials and methods

All chromatographic experiments were performed using ÄKTA™ explorer 10 with UNICORN™ 4.11 software (Amersham Biosciences, Uppsala, Sweden). E. coli TG1α cells were transfected with a 6125 bp derivative of pUC19 using a well-established protocol (Sambrook and Russel, 2001). Clarified alkaline lysate was prepared according to the protocol described by Horn et al. (1995).

Results

By group separation of clarified cell lysate in the presence of buffer containing 2 M (NH4)2SO4, plasmid DNA could be clearly separated from RNA by size exclusion chromatography on Sepharose 6 Fast Flow (Fig. 1). This procedure yielded a partially purified plasmid preparation containing open circular and supercoiled forms of plasmid DNA, with very little contaminating RNA. If the salt concentration was too low, however, the plasmid and RNA elution peaks were congruent. Unlike traditional gel

Discussion

Because of similarities in their polymeric composition, RNA, genomic DNA, and isoforms of plasmid DNA are difficult to separate. However, in the presence of water-structuring salts, these molecules are compacted differently and undergo conformational changes that can be exploited for their purification. In this study, we have identified aromatic thioether ligand structures that, when coupled to a chromatographic support, have the ability to separate supercoiled and open circular forms of

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