Sodium citrate and potassium phosphate as alternative adsorption buffers in hydrophobic and aromatic thiophilic chromatographic purification of plasmid DNA from neutralized lysate

dc.creatorBonturi, N
dc.creatorRadke, VSCO
dc.creatorBueno, SMA
dc.creatorFreitas, S
dc.creatorAzzoni, AR
dc.creatorMiranda, EA
dc.date2013
dc.date36951
dc.date2014-07-30T18:43:03Z
dc.date2015-11-26T16:45:19Z
dc.date2014-07-30T18:43:03Z
dc.date2015-11-26T16:45:19Z
dc.date.accessioned2018-03-28T23:30:53Z
dc.date.available2018-03-28T23:30:53Z
dc.identifierJournal Of Chromatography B-analytical Technologies In The Biomedical And Life Sciences. Elsevier Science Bv, v. 919, n. 67, n. 74, 2013.
dc.identifier1570-0232
dc.identifierWOS:000316043300010
dc.identifier10.1016/j.jchromb.2013.01.010
dc.identifierhttp://www.repositorio.unicamp.br/jspui/handle/REPOSIP/71736
dc.identifierhttp://repositorio.unicamp.br/jspui/handle/REPOSIP/71736
dc.identifier.urihttp://repositorioslatinoamericanos.uchile.cl/handle/2250/1274182
dc.descriptionFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
dc.descriptionCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
dc.descriptionConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
dc.descriptionThe number of studies on gene therapy using plasmid vectors (pDNA) has increased in recent years. As a result, the demand for preparations of pDNA in compliance with recommendations of regulatory agencies (EMEA, FDA) has also increased. Plasmid DNA is often obtained through fermentation of transformed Escherichia coli and purification by a series of unit operations, including chromatography. Hydrophobic interaction chromatography (HIC) and thiophilic aromatic chromatography (TAC), both using ammonium sulfate buffers, are commonly employed with success. This work was aimed at studying the feasibility of utilizing alternative salts in the purification of pDNA from neutralized lysate with phenyl-agarose (HIC) and mercaptopyrimidine-agarose (TAC) adsorbents. Their selectivity toward sc pDNA was evaluated through adsorption studies using 1.5 mol/L sodium citrate and 2.0 mol/L potassium phosphate as adsorption buffers. Chromatography with mercaptopyrimidine-agarose adsorbent and 1.5 mol/L sodium citrate was able to recover 91.1% of the pDNA with over 99.0% removal of gDNA and endotoxin. This represents a potential alternative for the primary recovery of sc pDNA. However, the most promising result was obtained using 2.0 mol/L potassium phosphate buffer and a mercaptopyrimidine-agarose column. In a single chromatographic step, this latter buffer/adsorbent system recovered 68.5% of the pDNA with 98.8% purity in accordance with the recommendations of regulatory agencies with regard to RNA and endotoxin impurity. (C) 2013 Elsevier B.V. All rights reserved.
dc.description919
dc.description67
dc.description74
dc.descriptionFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
dc.descriptionCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
dc.descriptionConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
dc.descriptionFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
dc.descriptionCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
dc.descriptionConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
dc.languageen
dc.publisherElsevier Science Bv
dc.publisherAmsterdam
dc.publisherHolanda
dc.relationJournal Of Chromatography B-analytical Technologies In The Biomedical And Life Sciences
dc.relationJ. Chromatogr. B
dc.rightsfechado
dc.rightshttp://www.elsevier.com/about/open-access/open-access-policies/article-posting-policy
dc.sourceWeb of Science
dc.subjectAromatic thiophilic chromatography
dc.subjectHydrophobic interaction chromatography
dc.subjectpDNA
dc.subjectNeutralized lysate
dc.subjectLiquid-liquid Equilibria
dc.subjectAqueous 2-phase Systems
dc.subjectGene-therapy
dc.subjectSelective Purification
dc.subjectWaste-water
dc.subjectVaccine
dc.subjectPerformance
dc.subjectRecovery
dc.subjectRemoval
dc.subjectImpact
dc.titleSodium citrate and potassium phosphate as alternative adsorption buffers in hydrophobic and aromatic thiophilic chromatographic purification of plasmid DNA from neutralized lysate
dc.typeArtículos de revistas


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