Purification of plasmid DNA with a new type of anion-exchange beads having a non-charged surface

Gustavsson, Per-Erik; Lemmens, Raf; Nyhammar, Tomas; Busson, P.; Larsson, Per‐Olof

doi:10.1016/j.chroma.2004.03.050

Cited by 43 publications

(29 citation statements)

References 18 publications

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“…Ljunglof et al [4] showed by confocal microscopy that media designed primarily for protein purification act as nonporous beads for pDNA, i. e. molecules are too big to diffuse into the pores and hence bind as outer layer only. Gustavsson et al [5] on the other hand took advantage of this drawback and introduced a flow-through purification protocol for pDNA on anion exchange chromatography (AEC) with a noncharged outer surface. Nonetheless the design of alternative media is definitively in great demand.…”

Section: Introductionmentioning

confidence: 99%

Adsorption of plasmid DNA on anion exchange chromatography media

Tarmann

Jungbauer²

2008

J of Separation Science

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Original PaperAdsorption of plasmid DNA on anion exchange chromatography media Anion exchange chromatography (AEC) is a useful and effective tool for DNA purification, but due to average pore sizes between 40 and 100 nm most AEC resins lack truly useful binding capacities for plasmid DNA (pDNA). Equilibrium binding capacities and uptake kinetics of AEC media including conventional media (Source 30 Q, Q Sepharose HP), a polymer grafted medium (Fractogel EMD DEAE (M)), media with large pores (Celbeads DEAE, PL SAX 4000 30 lm) and a monolithic medium (CIM-DEAE) were investigated by batch uptake or shallow bed experiments at two salt concentrations. Theoretical and experimental binding capacities suggest that the shape of the pDNA molecule can be described by a rod with a length to diameter ratio of 20:1 and that the molecule binds in upright position. The arrangement of DNA like a brush at the surface can be considered as entropy driven, kind of selfassembly process which is inherent to highly and uniformly charged DNA molecules. The initial phase of adsorption is very fast and levels off, associated with a change in mass transfer mechanism. Feed concentrations higher than 0.1 mg/mL pDNA pronounce this effect. Monolithic media showed the fastest adsorption rate and highest binding capacity with 13 mg pDNA per mL.

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Section: Introductionmentioning

confidence: 99%

Adsorption of plasmid DNA on anion exchange chromatography media

Tarmann

Jungbauer²

2008

J of Separation Science

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“…The present study concerns the simplest multi-layered multi-functional support design one can envisage, 30 namely one featuring just two differently functionalised layers -an inert outer size excluding layer and inner ion exchange functionalised core. The benefits of bi-layered size exclusion chromatography -ion exchange chromatography (SEC-IEC) beaded support designs have been clearly demonstrated in the context of 'nanoplex' purification [7,8], fluidised bed separation of organic acids [9,10] and expanded bed adsorption of proteins [11,12]. The important findings from these studies, inherent flaws in the methods employed thus far to manufacture bi-layered SEC-IEC hybrids, and identification of a simple and effective solution 5…”

mentioning

confidence: 97%

“…Starting from an SEC matrix with a nucleic acid exclusion limit of 1000 bp, Gustavsson and co-workers [7] made a bi-functional restricted access matrix possessing a positively charged core (to adsorb large amounts of RNA and protein) and an inert outer layer to exclude pDNA from accessing the functionalised bead interior. The creation of the two layers within the matrix was achieved in an ingenious multi-step process, which relied on the 30 use of limiting concentrations of reactants and 'diffusion/reaction' balancing in the second step.…”

mentioning

confidence: 99%

“…Recently, it has been demonstrated that much greater productivity could be realised if the SEC and AEC operations are 'passively' combined in a single chromatographic operation, employing a new type of multi-functional chromatography material (known as the lid bead) 25 [7,8]. Starting from an SEC matrix with a nucleic acid exclusion limit of 1000 bp, Gustavsson and co-workers [7] made a bi-functional restricted access matrix possessing a positively charged core (to adsorb large amounts of RNA and protein) and an inert outer layer to exclude pDNA from accessing the functionalised bead interior.…”

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confidence: 99%

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Surface modification of chromatography adsorbents by low temperature low pressure plasma

Arpanaei¹,

Winther‐Jensen²,

Theodosiou³

et al. 2010

Journal of Chromatography A

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“…This new type of chromatography matrix combines size exclusion and anion-exchange principle. In comparison to SEC this method has a much higher volumetric capacity and delivers the plasmids in a concentrated form (Gustavsson et al 2004). Indeed, this strategy is referred as a great promise as single-unit primary recovery step for the purification and concentration of pDNA from clarified alkaline lysates.…”

Section: Anion-exchange Chromatographymentioning

confidence: 99%

Biomedical application of plasmid DNA in gene therapy: A new challenge for chromatography

Sousa

Passarinha²,

Queiroz³

2009

Biotechnology and Genetic Engineering Reviews

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Gene therapy and DNA vaccination are clinical fields gradually emerging in the last few decades, in particular after the discovery of some gene-related diseases. The increased relevance of biomedical applications of plasmid DNA (pDNA) to induce therapeutic effects has had a great impact on biopharmaceutical research and industry. Although there are several steps involved in the pDNA manufacturing process, the several unit operations must be designed and integrated into a global process. After the plasmid has been designed according to the requirements for clinical administeration to humans, it is biosynthesised mainly by an E. coli host. The overriding priority of the production process is to improve plasmid quantity -the production conditions need to be optimised to guarantee pDNA stability and biological activity.The complexity and diversity of biomolecules present on the pDNA-containing extracts represent the main concern and limitation to achieve pure and biologically active pDNA. There has been a recent intenstification of the improvement of existing purification procedures or the establishment of novel schemes for plasmid purification.

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Purification of plasmid DNA with a new type of anion-exchange beads having a non-charged surface

Cited by 43 publications

References 18 publications

Adsorption of plasmid DNA on anion exchange chromatography media

Adsorption of plasmid DNA on anion exchange chromatography media

Surface modification of chromatography adsorbents by low temperature low pressure plasma

Biomedical application of plasmid DNA in gene therapy: A new challenge for chromatography

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