Supercoiled plasmid DNA: selective purification by thiophilic/aromatic adsorption

Lemmens, Raf; Olsson, Urban; Nyhammar, Tomas; Stadler, Joachim

doi:10.1016/s1570-0232(02)00805-x

Cited by 80 publications

(56 citation statements)

References 22 publications

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“…Zorbax GF250 is a SEC media that requires RNase pre-digestion, which is time and energy consuming [46]. Out of all the options, Sepharose 6 Fast Flow is currently considered the best media with better pDNA selectivity over RNA and experiences compacting effect from ammonium sulphate elution buffer [56]. Agarose gel electrophoresis and restriction analysis are then used for pDNA purity evaluation [34].…”

Section: Size Exclusion Chromatographymentioning

confidence: 99%

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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Section: Size Exclusion Chromatographymentioning

confidence: 99%

Chromatographic Removal of Endotoxins: A Bioprocess Engineer's Perspective

Ongkudon

Chew

Liu

et al. 2012

ISRN Chromatography

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“…Because complete separation of plasmid DNA and RNA is difficult to achieve with these methods under preparative conditions, they are preferably used for the depletion of residual contaminants and product concentration in subsequent downstream processing. Size exclusion chromatography allows the separation of plasmid DNA and RNA under suitable conditions [24,45]. However, this method suffers from its low capacity, long process time, and its inability to concentrate the product.…”

Section: Separation Of Plasmid Dna and Rnamentioning

confidence: 99%

Downstream Processing of Plasmid DNA for Gene Therapy and Genetic Vaccination

Voss

2008

Chem Eng & Technol

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Plasmid DNA is used as a cloning vector to deliver recombinant genetic information into microorganisms. Since the 1990s, this principle has also been applied for the delivery of therapeutic genes in gene therapy and genetic vaccination. This non-viral gene delivery is afflicted with fewer safety concerns in comparison to viral systems. Processes for the production of high-quality plasmid DNA at multiand kilogram scale are necessary to meet the needs of clinical trials as well as future therapeutics. Cell disruption, the separation of structurally-related impurities and analytical techniques for process and quality control are the main challenges for bioengineering. This review summarizes the development in these fields over the past recent years.

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“…15 Thus, the plasmid would be recovered as a flow-through fraction before the breakthrough of other impurities (proteins and RNA). To detect the breakthrough volume of the impurities, 20 mL of the feedstock was loaded to the column through the Superloop 50 mL (Amersham Biosciences) at 1 mL/min.…”

Section: Plasmid Purification By Chromatographymentioning

confidence: 99%

“…Lemmens et al 15 have found that supercoiled plasmid DNA could be isolated from other isoforms of plasmid DNA by Sepharose 6FF coupled with 2-mercaptopyridine in the presence of ammonium sulfate. However, this was not realized by the present column when adjusting the concentration of ammonium sulfate in the loading buffer.…”

Section: Separation Of Pcdna3 By Chromatographymentioning

confidence: 99%

“…It has been reported that mercaptopyridine ligand showed favorable performance in plasmid DNA purification modulated by high concentrations of lyotropic salt. 15 Because the mercaptopyridine ligand binds proteins and RNA preferentially, we have performed the purification by the flow-through mode of pDNA. Because the molecular masses of proteins and RNA are much smaller than pDNA, higher binding capacities of the adsorbent for these relatively low molecular-mass substances would be possible.…”

Section: Introductionmentioning

confidence: 99%

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Biporous polymeric microspheres coupled with mercaptopyridine for rapid chromatographic purification of plasmid DNA

Dong

Sun

2007

J of Applied Polymer Sci

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A biporous absorbent coupled with mercaptopyridine was synthesized for the purification of plasmid DNA. Analyses by scanning electron microscopy and mercury intrusion porosimetry revealed that the matrix contained two families of pores, i.e., micropores and superpores. The superpores provided not only convective flow channels for the mobile phase, but also a large surface for biomacromolecules binding. So the chromatographic process can be operated at high flow rate with high column efficiency and low backpressure as identified on a 2-mL column. When 10 mL crude feedstock containing 3 mg of plasmid (5.4 kb pcDNA3) was loaded at a flow rate as high as 20 cm/min, the separation was finished in 10 min, and the plasmid was completely recovered with undetectable impurities of nucleic acids and proteins. The productivity was determined to be 9.0 g/L h, comparable to the pDNA productivity obtained using the commercial column. These results indicate that the biporous medium is promising for high-throughput purification of plasmid DNA.

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Supercoiled plasmid DNA: selective purification by thiophilic/aromatic adsorption

Cited by 80 publications

References 22 publications

Chromatographic Removal of Endotoxins: A Bioprocess Engineer's Perspective

Chromatographic Removal of Endotoxins: A Bioprocess Engineer's Perspective

Downstream Processing of Plasmid DNA for Gene Therapy and Genetic Vaccination

Biporous polymeric microspheres coupled with mercaptopyridine for rapid chromatographic purification of plasmid DNA

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