Application of short monolithic columns for fast purification of plasmid DNA

Branović, Karmen; Forčić, Dubravko; Ivančić, Jelena; Štrancar, Aleš; Barut, Miloš; Gulija, Tanja Košutić; Zgorelec, Renata; Mažuran, Renata

doi:10.1016/j.jchromb.2003.11.035

Cited by 72 publications

(32 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…By comparing biophysical characteristics such as molecular diffusivity (D) and molecular weight (Mw) of proteins and pDNA it is obvious that the use of conventional media derives from a lack of attractive alternatives. D values being a magnitude of 10 smaller than proteins and Mw in the range of 10 6 Da make conventional media suffer from low capacity and slow mass transfer [3]. 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.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Adsorption of plasmid DNA on anion exchange chromatography media

Tarmann

Jungbauer²

2008

J of Separation Science

View full text Add to dashboard Cite

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.

show abstract

Section: Introductionmentioning

confidence: 99%

“…Nonetheless the design of alternative media is definitively in great demand. By now monolithic supports are well-established alternatives for purification of large biomolecules [6,7]. Monoliths are continuous stationary phases with a very wide macropore structure providing convective flow inside the pores [8].…”

Section: Introductionmentioning

confidence: 99%

Adsorption of plasmid DNA on anion exchange chromatography media

Tarmann

Jungbauer²

2008

J of Separation Science

View full text Add to dashboard Cite

show abstract

“…The pDNA was purified on a DEAE CIM disk from BIA Separation (Ljubljana, Slovenia) as previously described. 27 The concentration and purity of the pDNA was determined by the ratio A260/280 and by agarose gel electrophoresis.…”

Section: Materials and Methods Chemicalsmentioning

confidence: 99%

Influence of charge ratio of liposome/DNA complexes on their size after extrusion and transfection efficiency

Brgles¹,

Šantak²,

Halassy³

et al. 2012

IJN

Self Cite

View full text Add to dashboard Cite

Background: Physicochemical characteristics of liposome/DNA complexes influence transfection efficiency and affect each other in a very intricate way. The result of this is discrepancies in conclusions drawn about the individual influence of each one. Methods: Aiming to elucidate the influence of liposome/DNA charge ratio and size on transfection efficiency and on each other, we used liposome/DNA complexes with charge ratio (+/−) in the range of 1-50 and extruded through membranes of 400, 200, and 100 nm. Plasmid DNA encoding green fluorescent protein was used to measure transfection efficiency by flow cytometry. Sizes of liposome/DNA complexes were measured by dynamic light scattering. Results: Liposome size was reduced after extrusion but this was mainly driven by the charge ratio and not by the size of the membrane pores. Reduction of complex size at each charge ratio positively correlated with transfection efficiency. When the size of the complexes was approximately constant, increasing the charge ratio was found to promote transfection efficiency. Cationic lipid N-(1-(2,3-dioleoyloxy)propyl)N,N,N trimethylammonium chloride was used for modulation of positive charge and a cytotoxicity test showed that increasing its amount increases cytotoxicity. Conclusion: It can be concluded that charge ratio dictates the size of the complex whereas overall size reduction and higher charge ratios promote transfection efficiency in vitro.

show abstract

“…In fact, the application of monolithic columns to purify pDNA is an emerging area and can represent some technical advantages, because this large macromolecule presents very different biophysical properties than proteins. Together with monoliths (Bencina et al 2004;Branovic et al 2004;Jungbauer and Hahn 2004;Urthaler et al 2005), other new supports have been developed to overcome the diffusion limitation as well as to improve the binding capacity of the support for the target molecules. Such superporous supports (Gustavsson et al 1999;Tiainen et al 2007b), and adsorptive membranes (Giovannini et al 1998;Teeters et al 2003) are advanced approaches that have been shown to be able to partially solve the problems associated with low capacity.…”

Section: Chromatographic Perspectives Enhancing Pdna Purificationmentioning

confidence: 99%

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

Sousa

Passarinha²,

Queiroz³

2009

Biotechnology and Genetic Engineering Reviews

View full text Add to dashboard Cite

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.

show abstract

Application of short monolithic columns for fast purification of plasmid DNA

Cited by 72 publications

References 27 publications

Adsorption of plasmid DNA on anion exchange chromatography media

Adsorption of plasmid DNA on anion exchange chromatography media

Influence of charge ratio of liposome/DNA complexes on their size after extrusion and transfection efficiency

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

Contact Info

Product

Resources

About