Initial Screening of Poly(ethylene glycol) Amino Ligands for Affinity Purification of Plasmid DNA in Aqueous Two-Phase Systems

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,...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Nuno R. da Silva, Paula Jorge, José A. Martins, José A. Teixeira, João C. Marcos
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	aqueous two-phase systems affinity partition non-viral vectors plasmid DNA purification gene therapy DNA vaccines Science Q
Acceso en línea:	https://doaj.org/article/3933f99fe1864318b7a9707462897032
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:3933f99fe1864318b7a9707462897032
record_format	dspace
spelling	oai:doaj.org-article:3933f99fe1864318b7a97074628970322021-11-25T18:10:34ZInitial Screening of Poly(ethylene glycol) Amino Ligands for Affinity Purification of Plasmid DNA in Aqueous Two-Phase Systems10.3390/life111111382075-1729https://doaj.org/article/3933f99fe1864318b7a97074628970322021-10-01T00:00:00Zhttps://www.mdpi.com/2075-1729/11/11/1138https://doaj.org/toc/2075-1729Gene 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 (PEG–amine) or conjugation with positively charged amino acids (PEG–lysine, PEG–arginine, and PEG–histidine) were studied to increase the selectivity of PEG–dextran 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 PEG–dextran 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.Nuno R. da SilvaPaula JorgeJosé A. MartinsJosé A. TeixeiraJoão C. MarcosMDPI AGarticleaqueous two-phase systemsaffinity partitionnon-viral vectorsplasmid DNA purificationgene therapyDNA vaccinesScienceQENLife, Vol 11, Iss 1138, p 1138 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	aqueous two-phase systems affinity partition non-viral vectors plasmid DNA purification gene therapy DNA vaccines Science Q
spellingShingle	aqueous two-phase systems affinity partition non-viral vectors plasmid DNA purification gene therapy DNA vaccines Science Q Nuno R. da Silva Paula Jorge José A. Martins José A. Teixeira João C. Marcos Initial Screening of Poly(ethylene glycol) Amino Ligands for Affinity Purification of Plasmid DNA in Aqueous Two-Phase Systems
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 (PEG–amine) or conjugation with positively charged amino acids (PEG–lysine, PEG–arginine, and PEG–histidine) were studied to increase the selectivity of PEG–dextran 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 PEG–dextran 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.
format	article
author	Nuno R. da Silva Paula Jorge José A. Martins José A. Teixeira João C. Marcos
author_facet	Nuno R. da Silva Paula Jorge José A. Martins José A. Teixeira João C. Marcos
author_sort	Nuno R. da Silva
title	Initial Screening of Poly(ethylene glycol) Amino Ligands for Affinity Purification of Plasmid DNA in Aqueous Two-Phase Systems
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
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/3933f99fe1864318b7a9707462897032
work_keys_str_mv	AT nunordasilva initialscreeningofpolyethyleneglycolaminoligandsforaffinitypurificationofplasmiddnainaqueoustwophasesystems AT paulajorge initialscreeningofpolyethyleneglycolaminoligandsforaffinitypurificationofplasmiddnainaqueoustwophasesystems AT joseamartins initialscreeningofpolyethyleneglycolaminoligandsforaffinitypurificationofplasmiddnainaqueoustwophasesystems AT joseateixeira initialscreeningofpolyethyleneglycolaminoligandsforaffinitypurificationofplasmiddnainaqueoustwophasesystems AT joaocmarcos initialscreeningofpolyethyleneglycolaminoligandsforaffinitypurificationofplasmiddnainaqueoustwophasesystems
_version_	1718411491327082496

Initial Screening of Poly(ethylene glycol) Amino Ligands for Affinity Purification of Plasmid DNA in Aqueous Two-Phase Systems

Ejemplares similares