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Minicircle DNA purification: Performance of chromatographic monoliths bearing lysine and cadaverine ligands

2019Journal article Restricted access
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dc.contributor.authorAlmeida, Ana Margarida
dc.contributor.authorQueiroz, João
dc.contributor.authorSousa, Fani
dc.contributor.authorSousa, Ângela
dc.date.accessioned2020-01-10T10:01:03Z
dc.date.available2020-01-10T10:01:03Z
dc.date.issued2019
dc.description.abstractMinicircle DNA (mcDNA) technology is in the vanguard of vectors designed for gene therapy, since the absence of prokaryotic sequences confers to mcDNA higher biosafety in comparison to other DNA vectors. However, the presence of other isoforms and non-recombined parental molecules hampers the isolation of supercoiled (sc) mcDNA with the chromatographic methods already established for plasmid purification. In this work, two monolithic supports were modified with lysine and its decarboxylated derivative, cadaverine, to explore their performance in the sc mcDNA purification. Increasing NaCl gradients and different pH values (from 6 to 9) were tested in both modified monoliths. In general, cadaverine modified support established stronger interactions with mcDNA than lysine modified monolith, at acidic pH. For instance, at pH 6.0 the retention time for RNA and DNA molecules in lysine modified monolith was 11.58 and 14.59, respectively, while for cadaverine modified monolith was 20.32 and 27.12, respectively. The lysine modified monolith was able to successfully isolate sc mcDNA from the lysate sample. However, recovery yield was significantly sacrificed to guarantee high purity levels of sc mcDNA. The cadaverine modified monolith showed better selectivity than the previous monolith, achieving the successful sc mcDNA isolation from the lysate sample. The final sc mcDNA sample, obtained by the column that showed the best performance, was characterized by real-time PCR, presenting 98.4% purity and 78.6% recovery yield. The impurities content, namely genomic DNA, proteins and endotoxins, was found within the criteria established by regulatory agencies. Overall, a simple and practical chromatographic strategy to purify sc mcDNA was for the first time implemented by exploring a modified monolithic column, with no significant reduction on the purity and recovery and without resorting to backbone modification or specific enzymatic digestion. Such features will surely be crucial in the industrial scale-up of this chromatographic strategy since it will not be associated with significant cost-increase.pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.doi10.1016/j.jchromb.2019.04.024pt_PT
dc.identifier.urihttp://hdl.handle.net/10400.6/8188
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherElsevierpt_PT
dc.relationBiosynthesis and purification of minicircle DNA encoding pri-miR-375 for treatment of Human Papillomavirus infection
dc.relation.publisherversionhttps://www.sciencedirect.com/science/article/pii/S1570023218317008pt_PT
dc.subjectCadaverinept_PT
dc.subjectChromatographypt_PT
dc.subjectDNApt_PT
dc.subjectDNA, Superhelicalpt_PT
dc.subjectElectrophoresispt_PT
dc.subjectHydrogen-Ion Concentrationpt_PT
dc.subjectLysinept_PT
dc.titleMinicircle DNA purification: Performance of chromatographic monoliths bearing lysine and cadaverine ligandspt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardTitleBiosynthesis and purification of minicircle DNA encoding pri-miR-375 for treatment of Human Papillomavirus infection
oaire.awardURIinfo:eu-repo/grantAgreement/FCT//SFRH%2FBD%2F102284%2F2014/PT
oaire.citation.endPage16pt_PT
oaire.citation.startPage7pt_PT
oaire.citation.titleJournal of Chromatography Bpt_PT
oaire.citation.volume1118-1119pt_PT
person.familyNameAlmeida
person.familyNameQueiroz
person.familyNameSousa
person.familyNameSousa
person.givenNameAna Margarida
person.givenNameJoão
person.givenNameFani
person.givenNameÂngela
person.identifier817754
person.identifier.ciencia-id931E-B66D-E341
person.identifier.ciencia-id991D-2E13-A840
person.identifier.ciencia-idFB1F-08DE-23FE
person.identifier.orcid0000-0002-9313-6649
person.identifier.orcid0000-0002-3096-8325
person.identifier.orcid0000-0001-9996-2194
person.identifier.orcid0000-0001-9155-7581
person.identifier.ridL-3104-2014
person.identifier.ridA-2014-2017
person.identifier.scopus-author-id7003705645
person.identifier.scopus-author-id7005110268
person.identifier.scopus-author-id24330268800
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.nameFundação para a Ciência e a Tecnologia
rcaap.embargofctCopyright cedido à editora no momento da publicaçãopt_PT
rcaap.rightsclosedAccesspt_PT
rcaap.typearticlept_PT
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