Plasmid purification by hydrophobic interaction chromatography using sodium citrate in the mobile phase

Freitas, Sindélia; Santos, J.P.; Prazeres, D.M.F.

doi:10.1016/j.seppur.2008.04.001

Cited by 33 publications

(18 citation statements)

References 29 publications

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“…Plasmid concentration and purity were determined by hydrophobic interaction, high performance liquid chromatography (HIC-HPLC), as described previously [30,31]. Briefly, the HIC source 15 PHE PE column was connected to a Ä kta Purifier FPLC system from Amersham Biosciences (Uppsala, Sweden) and equilibrated with 1.5 M ammonium sulfate in Tris-HCl 10 mM, pH 8.0.…”

Section: Hydrophobic Interaction Hplcmentioning

confidence: 99%

Purification of plasmid DNA using tangential flow filtration and tandem anion-exchange membrane chromatography

Guerrero-Germán

Prazeres

Guzmán

et al. 2008

Bioprocess Biosyst Eng

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A new bioprocess using mainly membrane operations to obtain purified plasmid DNA from Escherechia coli ferments was developed. The intermediate recovery and purification of the plasmid DNA in cell lysate was conducted using hollow-fiber tangential filtration and tandem anion-exchange membrane chromatography. The purity of the solutions of plasmid DNA obtained during each process stage was investigated. The results show that more than 97% of RNA in the lysate was removed during the process operations and that the plasmid DNA solution purity increased 28-fold. One of the main characteristics of the developed process is to avoid the use of large quantities of precipitating agents such as salts or alcohols. A better understanding of membrane-based technology for the purification of plasmid DNA from clarified E. coli lysate was developed in this research. The convenience of anion-exchange membranes, configured in ready-to-use devices can further simplify large-scale plasmid purification strategies.

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Section: Hydrophobic Interaction Hplcmentioning

confidence: 99%

Purification of plasmid DNA using tangential flow filtration and tandem anion-exchange membrane chromatography

Guerrero-Germán

Prazeres

Guzmán

et al. 2008

Bioprocess Biosyst Eng

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“…Immobilized hydrophobic ligands and reverse-phase chromatography (RPC) Interact with non-polar protein surfaces through van der Waals forces for high endotoxin removal [49,50] combination of electrostatic, hydrophobic and hydrogen bond interactions are present in the affinity chromatography [36]. Bound substances can be recovered or removed through desorption process.…”

Section: Affinity-basedmentioning

confidence: 99%

“…Hydrophobic interaction chromatography (HIC) explores differences in hydrophobicity for plasmid purification and captures pDNA under high-salt conditions due to the predominant hydrophilic feature of pDNA [49,60]. Immobilized hydrophobic ligands interact with nonpolar protein surfaces through van der Waals forces for high endotoxin removal [49]. Protein and endotoxin are adsorbed onto the ligands and later separated using salt addition based on gradient elution [35].…”

Section: Hydrophobic Interactionmentioning

confidence: 99%

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Chromatographic Removal of Endotoxins: A Bioprocess Engineer's Perspective

Ongkudon

Chew

Liu

et al. 2012

ISRN Chromatography

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Gram-negative bacteria are widely used for the production of gene-based products such as DNA vaccines and bio-drugs, where endotoxin contamination can occur at any point within the process and its removal is of great concern. In this article, we review the structures of endotoxin and the effects that it causes in vivo. The endotoxin removal strategies are also discussed in the light of the different interaction mechanisms involved between endotoxins and bioproducts particularly plasmid DNA and proteins. For most cases, endotoxin removal is favoured at a highly ionic or acidic condition. Various removal methods particularly chromatographybased techniques are covered in this article according to the relevant applications.

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“…Chromatographic processes are usually preferred to remove impurities and to separate supercoiled pDNA from other isoforms. Affinity chromatography (AC), anion-exchange chromatography (AEC), reversed-phase chromatography (RP), size-exclusion chromatography (SEC), singly or combined, may be employed (6)(7)(8)(9)(10)(11)(12)(13). The non-selectivity of adsorbents leading to the co-elution of impurities in AEC, and the toxicity of organic solvents used in RP, are the disadvantages of these methods.…”

Section: Introductionmentioning

confidence: 99%

Magnetic Nanoparticles for Plasmid DNA Purification through Hydrophobic Interaction Chromatography

Üzek

Özkara

Güngüneş

et al. 2014

Separation Science and Technology

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Magnetic poly(2-hydroxyethyl methacrylate-co-N-methacryloyl-L-phenyl alanine), p(HEMA-co-MAPA) nanoparticles were produced by emulsion polymerization technique. The nanoparticles were characterized by zetasizer, atomic force microscopy (AFM), transmission electron microscopy (TEM), electron spin resonance spectroscopy (ESR), and Fourier transformed infrared spectroscopy (FTIR) techniques. Experimental conditions were optimized for DNA adsorption in aqueous media. The effects of several factors (i.e., temperature, pH, equilibrium concentration of DNA, salt type, and ionic strength) on adsorption capacity were examined in batch studies. The maximum DNA adsorption capacity was 211.3 mg/g for nanoparticles consisting of magnetite. The adsorption isotherms and kinetics were also examined. The Langmuir model and pseudo second-order kinetics are the best fitted to the data. Following the ten adsorption-desorption cycles, the decrease in adsorption capacity was only 12%. Finally, plasmid DNA was purified from E.coli lysate by magnetite nanoparticles consisting of MAPA. The purity and molecular mass of purified plasmid DNA were determined by the use of agarose gel electrophoresis resulting in 7000 base pairs for molecular mass and OC pDNA form following elution.

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Plasmid purification by hydrophobic interaction chromatography using sodium citrate in the mobile phase

Cited by 33 publications

References 29 publications

Purification of plasmid DNA using tangential flow filtration and tandem anion-exchange membrane chromatography

Purification of plasmid DNA using tangential flow filtration and tandem anion-exchange membrane chromatography

Chromatographic Removal of Endotoxins: A Bioprocess Engineer's Perspective

Magnetic Nanoparticles for Plasmid DNA Purification through Hydrophobic Interaction Chromatography

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