Initial screening of poly(ethylene glycol) amino ligands for affinity purification of plasmid DNA in aqueous two-phase systems
Autor(a) principal: | |
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Data de Publicação: | 2021 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | https://hdl.handle.net/1822/74590 |
Resumo: | Gene therapy and DNA vaccination are among the most expected biotechnological and medical advances for the coming years. However, the lack of cost-effective large-scale production and purification of pharmaceutical-grade plasmid DNA (pDNA) still hampers their wide application. Downstream processing, which is mainly chromatography-based, of pDNA remains the key manufacturing step. Despite its high resolution, the scaling-up of chromatography is usually difficult and presents low capacity, resulting in low yields. Alternative methods that are based on aqueous two-phase systems (ATPSs) have been studied. Although higher yields may be obtained, its selectivity is often low. In this work, modified polymers based on poly(ethylene glycol) (PEG) derivatisation with amino groups (PEGamine) or conjugation with positively charged amino acids (PEGlysine, PEGarginine, and PEGhistidine) were studied to increase the selectivity of PEGdextran systems towards the partition of a model plasmid. A two-step strategy was employed to obtain suitable pure formulations of pDNA. In the first step, a PEGdextran system with the addition of the affinity ligand was used with the recovery of the pDNA in the PEG-rich phase. Then, the pDNA was re-extracted to an ammonium-sulphate-rich phase in the second step. After removing the salt, this method yielded a purified preparation of pDNA without RNA and protein contamination. |
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Initial screening of poly(ethylene glycol) amino ligands for affinity purification of plasmid DNA in aqueous two-phase systemsAqueous two-phase systemsAffinity partitionNon-viral vectorsPlasmid DNA purificationGene therapyDNA vaccinesScience & TechnologyGene therapy and DNA vaccination are among the most expected biotechnological and medical advances for the coming years. However, the lack of cost-effective large-scale production and purification of pharmaceutical-grade plasmid DNA (pDNA) still hampers their wide application. Downstream processing, which is mainly chromatography-based, of pDNA remains the key manufacturing step. Despite its high resolution, the scaling-up of chromatography is usually difficult and presents low capacity, resulting in low yields. Alternative methods that are based on aqueous two-phase systems (ATPSs) have been studied. Although higher yields may be obtained, its selectivity is often low. In this work, modified polymers based on poly(ethylene glycol) (PEG) derivatisation with amino groups (PEGamine) or conjugation with positively charged amino acids (PEGlysine, PEGarginine, and PEGhistidine) were studied to increase the selectivity of PEGdextran systems towards the partition of a model plasmid. A two-step strategy was employed to obtain suitable pure formulations of pDNA. In the first step, a PEGdextran system with the addition of the affinity ligand was used with the recovery of the pDNA in the PEG-rich phase. Then, the pDNA was re-extracted to an ammonium-sulphate-rich phase in the second step. After removing the salt, this method yielded a purified preparation of pDNA without RNA and protein contamination.Thanks are due to the Fundação para a Ciência e Tecnologia (FCT, Portugal) for financial support to the NMR Portuguese network (PTNMR, Bruker Avance III 400-Univ. Minho) and strategic funding for CQUM (UID/QUI/00686/2020).info:eu-repo/semantics/publishedVersionMultidisciplinary Digital Publishing Institute (MDPI)Universidade do MinhoSilva, Nuno Miguel Sampaio Ribeiro MagalhãesJorge, Paula Alexandra SilvaMartins, José A.Teixeira, J. A.Marcos, João C.20212021-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttps://hdl.handle.net/1822/74590engSilva, Nuno R. da; Jorge, Paula; Martins, José A.; Teixeira, José A.; Marcos, João C., Initial screening of poly(ethylene glycol) amino ligands for affinity purification of plasmid DNA in aqueous two-phase systems. Life, 11(11), 1138, 20210024-301910.3390/life11111138https://www.mdpi.com/2075-1729/11/11/1138info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-07-21T12:46:56ZPortal AgregadorONG |
dc.title.none.fl_str_mv |
Initial screening of poly(ethylene glycol) amino ligands for affinity purification of plasmid DNA in aqueous two-phase systems |
title |
Initial screening of poly(ethylene glycol) amino ligands for affinity purification of plasmid DNA in aqueous two-phase systems |
spellingShingle |
Initial screening of poly(ethylene glycol) amino ligands for affinity purification of plasmid DNA in aqueous two-phase systems Silva, Nuno Miguel Sampaio Ribeiro Magalhães Aqueous two-phase systems Affinity partition Non-viral vectors Plasmid DNA purification Gene therapy DNA vaccines Science & Technology |
title_short |
Initial screening of poly(ethylene glycol) amino ligands for affinity purification of plasmid DNA in aqueous two-phase systems |
title_full |
Initial screening of poly(ethylene glycol) amino ligands for affinity purification of plasmid DNA in aqueous two-phase systems |
title_fullStr |
Initial screening of poly(ethylene glycol) amino ligands for affinity purification of plasmid DNA in aqueous two-phase systems |
title_full_unstemmed |
Initial screening of poly(ethylene glycol) amino ligands for affinity purification of plasmid DNA in aqueous two-phase systems |
title_sort |
Initial screening of poly(ethylene glycol) amino ligands for affinity purification of plasmid DNA in aqueous two-phase systems |
author |
Silva, Nuno Miguel Sampaio Ribeiro Magalhães |
author_facet |
Silva, Nuno Miguel Sampaio Ribeiro Magalhães Jorge, Paula Alexandra Silva Martins, José A. Teixeira, J. A. Marcos, João C. |
author_role |
author |
author2 |
Jorge, Paula Alexandra Silva Martins, José A. Teixeira, J. A. Marcos, João C. |
author2_role |
author author author author |
dc.contributor.none.fl_str_mv |
Universidade do Minho |
dc.contributor.author.fl_str_mv |
Silva, Nuno Miguel Sampaio Ribeiro Magalhães Jorge, Paula Alexandra Silva Martins, José A. Teixeira, J. A. Marcos, João C. |
dc.subject.por.fl_str_mv |
Aqueous two-phase systems Affinity partition Non-viral vectors Plasmid DNA purification Gene therapy DNA vaccines Science & Technology |
topic |
Aqueous two-phase systems Affinity partition Non-viral vectors Plasmid DNA purification Gene therapy DNA vaccines Science & Technology |
description |
Gene therapy and DNA vaccination are among the most expected biotechnological and medical advances for the coming years. However, the lack of cost-effective large-scale production and purification of pharmaceutical-grade plasmid DNA (pDNA) still hampers their wide application. Downstream processing, which is mainly chromatography-based, of pDNA remains the key manufacturing step. Despite its high resolution, the scaling-up of chromatography is usually difficult and presents low capacity, resulting in low yields. Alternative methods that are based on aqueous two-phase systems (ATPSs) have been studied. Although higher yields may be obtained, its selectivity is often low. In this work, modified polymers based on poly(ethylene glycol) (PEG) derivatisation with amino groups (PEGamine) or conjugation with positively charged amino acids (PEGlysine, PEGarginine, and PEGhistidine) were studied to increase the selectivity of PEGdextran systems towards the partition of a model plasmid. A two-step strategy was employed to obtain suitable pure formulations of pDNA. In the first step, a PEGdextran system with the addition of the affinity ligand was used with the recovery of the pDNA in the PEG-rich phase. Then, the pDNA was re-extracted to an ammonium-sulphate-rich phase in the second step. After removing the salt, this method yielded a purified preparation of pDNA without RNA and protein contamination. |
publishDate |
2021 |
dc.date.none.fl_str_mv |
2021 2021-01-01T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://hdl.handle.net/1822/74590 |
url |
https://hdl.handle.net/1822/74590 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Silva, Nuno R. da; Jorge, Paula; Martins, José A.; Teixeira, José A.; Marcos, João C., Initial screening of poly(ethylene glycol) amino ligands for affinity purification of plasmid DNA in aqueous two-phase systems. Life, 11(11), 1138, 2021 0024-3019 10.3390/life11111138 https://www.mdpi.com/2075-1729/11/11/1138 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Multidisciplinary Digital Publishing Institute (MDPI) |
publisher.none.fl_str_mv |
Multidisciplinary Digital Publishing Institute (MDPI) |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
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repository.mail.fl_str_mv |
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1777303841802289152 |