Evaluation of novel chemistries on porous supports for plasmid DNA polishing chromatography
Autor(a) principal: | |
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Data de Publicação: | 2023 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10400.6/13622 |
Resumo: | DNA-based therapeutics are one of the main applications aiming to find a course of treatment for hereditary diseases as well as cancer. They provide additional benefits when compared to traditional therapeutics. Plasmid DNA (pDNA) is one of the most utilized species of DNA-based therapeutics due to its ability to induce significant expression of a trans-gene and a comparably easy manufacturing process, without any of the disadvantages viral vectors show. This form of DNA presents linear, open circle and supercoiled isoforms, while being possible to switch between them, based on the amount and location of nicks in the sequence. The supercoiled isoform is the most biologically significant one since it allows for higher efficiencies of trans-gene expression. In order to use pDNA for human treatment, it needs to be purified so that the final product consists of a homogeneous supercoiled pDNA pool. To obtain this, a chromatography process with a combination of several interactions presents the best course of action, which is demonstrated by multiple products currently on the market. Nevertheless, the existing supports that are focused on the chromatographic step specific for the isolation of the relevant pDNA isoform (polishing step) do not present the best performance characteristics, both in terms of the purity obtained and the yield. Therefore, the present work aims to develop a novel polishing chromatographic material to produce supercoiled pDNA with higher purity and recovery. To do this, firstly, static binding experiments were conducted to confirm the results previously obtained by another working collaboration. Then, the promising prototypes were subjected to dynamic binding conditions, with the objective of assessing their capabilities to isolate the supercoiled isoform. This was followed by the development of several new agmatine-based prototypes where elution buffer composition and pH were optimized to achieve the highest resolution and purity of supercoiled pDNA. Afterwards, a step-elution method and optimization were conducted with a 3rd generation agmatine prototype, since it presented the best results in terms of supercoiled purity. With the optimized method, a complex sample of RNA and pDNA was applied to the prototype to evaluate if it was capable of separating supercoiled pDNA from other isoforms and similar molecules. The results indicated that the agmatine prototype is efficient in the purification of supercoiled pDNA from complex samples, with a high degree of purification, and in accordance with the specifications of regulatory agencies. |
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Evaluation of novel chemistries on porous supports for plasmid DNA polishing chromatographyAgmatinaDna PlasmídicoIsoforma SuperenroladaSuporte de ResinaTerapêuticos de DnaDomínio/Área Científica::Engenharia e Tecnologia::BiotecnologiaDNA-based therapeutics are one of the main applications aiming to find a course of treatment for hereditary diseases as well as cancer. They provide additional benefits when compared to traditional therapeutics. Plasmid DNA (pDNA) is one of the most utilized species of DNA-based therapeutics due to its ability to induce significant expression of a trans-gene and a comparably easy manufacturing process, without any of the disadvantages viral vectors show. This form of DNA presents linear, open circle and supercoiled isoforms, while being possible to switch between them, based on the amount and location of nicks in the sequence. The supercoiled isoform is the most biologically significant one since it allows for higher efficiencies of trans-gene expression. In order to use pDNA for human treatment, it needs to be purified so that the final product consists of a homogeneous supercoiled pDNA pool. To obtain this, a chromatography process with a combination of several interactions presents the best course of action, which is demonstrated by multiple products currently on the market. Nevertheless, the existing supports that are focused on the chromatographic step specific for the isolation of the relevant pDNA isoform (polishing step) do not present the best performance characteristics, both in terms of the purity obtained and the yield. Therefore, the present work aims to develop a novel polishing chromatographic material to produce supercoiled pDNA with higher purity and recovery. To do this, firstly, static binding experiments were conducted to confirm the results previously obtained by another working collaboration. Then, the promising prototypes were subjected to dynamic binding conditions, with the objective of assessing their capabilities to isolate the supercoiled isoform. This was followed by the development of several new agmatine-based prototypes where elution buffer composition and pH were optimized to achieve the highest resolution and purity of supercoiled pDNA. Afterwards, a step-elution method and optimization were conducted with a 3rd generation agmatine prototype, since it presented the best results in terms of supercoiled purity. With the optimized method, a complex sample of RNA and pDNA was applied to the prototype to evaluate if it was capable of separating supercoiled pDNA from other isoforms and similar molecules. The results indicated that the agmatine prototype is efficient in the purification of supercoiled pDNA from complex samples, with a high degree of purification, and in accordance with the specifications of regulatory agencies.As terapias baseadas em DNA têm o objetivo, não só de encontrar formas de tratamento para doenças hereditárias, como também para o cancro e outras patologias, de forma a proporcionar benefícios adicionais, quando comparadas com as terapias tradicionais. O DNA plasmídico (pDNA) é uma das espécies mais utilizadas em terapias baseadas em DNA, devido à sua capacidade para induzir uma expressão significativa do transgene, através de um processo de produção simples, sem as desvantagens dos vetores virais. O pDNA possui as isoformas linear, circular aberta e superenrolada, dependendo da localização e número de cortes na cadeia. A isoforma superenrolada é biologicamente mais ativa, dado que proporciona maior eficiência na expressão do transgene. No entanto, para se utilizar o pDNA superenrolado no tratamento de humanos, esta isoforma tem de ser purificada de forma a obter um produto final que possua uma pool homogénea de pDNA superenrolado. Os processos cromatográficos de vários passos, com diferentes interações, têm sido a estratégia mais utilizada, existindo vários suportes atualmente no mercado, no entanto ainda não apresentam o melhor desempenho na etapa de polimento, tanto em termos grau de pureza obtido, como de rendimento. Deste modo, o presente trabalho tem como objetivo o desenvolvimento de um novo material cromatográfico para purificação de pDNA, com maior recuperação e pureza da isoforma superenrolada. Primeiramente, foram realizados ensaios de ligação estática em diferentes colunas cromatográficas e, posteriormente, os protótipos com os melhores resultados foram testados em condições de ligação dinâmica, com o objetivo de avaliar a sua capacidade de isolar a isoforma superenrolada. De seguida, diversos suportes cromatográficos foram preparados, tendo como base o protótipo de agmatina, e onde tanto a composição da solução de eluição, como o pH foram otimizados de forma a obter a maior resolução e elevada pureza da isoforma superenrolada. Finalmente, foi otimizado um método de eluição por passos utilizando o protótipo de agmatina de 3ra geração, dado que foi aquele que apresentou os melhores resultados, em termos de pureza da isoforma superenrolada. Com o método otimizado, o suporte de agmatina foi testado com uma amostra complexa com RNA e pDNA, de forma a avaliar a sua capacidade de separar de modo específico o pDNA superenrolado, na presença de outras moléculas com características semelhantes. Os resultados indicaram que o protótipo de agmatina é eficiente na purificação de amostras complexas, permitindo a obtenção de pDNA superenrolado, com um grau de purificação elevado e de acordo com as especificações das agências reguladoras.Rammo, OliverSousa, Fani Pereira deTomaz, Cândida Ascensão TeixeirauBibliorumLuís, Marco Ângelo2023-07-182023-06-129999-01-01T00:00:00Z2023-07-18T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10400.6/13622TID:203381882engmetadata only accessinfo: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:RCAAP2023-12-15T09:57:17Zoai:ubibliorum.ubi.pt:10400.6/13622Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T00:53:01.327722Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Evaluation of novel chemistries on porous supports for plasmid DNA polishing chromatography |
title |
Evaluation of novel chemistries on porous supports for plasmid DNA polishing chromatography |
spellingShingle |
Evaluation of novel chemistries on porous supports for plasmid DNA polishing chromatography Luís, Marco Ângelo Agmatina Dna Plasmídico Isoforma Superenrolada Suporte de Resina Terapêuticos de Dna Domínio/Área Científica::Engenharia e Tecnologia::Biotecnologia |
title_short |
Evaluation of novel chemistries on porous supports for plasmid DNA polishing chromatography |
title_full |
Evaluation of novel chemistries on porous supports for plasmid DNA polishing chromatography |
title_fullStr |
Evaluation of novel chemistries on porous supports for plasmid DNA polishing chromatography |
title_full_unstemmed |
Evaluation of novel chemistries on porous supports for plasmid DNA polishing chromatography |
title_sort |
Evaluation of novel chemistries on porous supports for plasmid DNA polishing chromatography |
author |
Luís, Marco Ângelo |
author_facet |
Luís, Marco Ângelo |
author_role |
author |
dc.contributor.none.fl_str_mv |
Rammo, Oliver Sousa, Fani Pereira de Tomaz, Cândida Ascensão Teixeira uBibliorum |
dc.contributor.author.fl_str_mv |
Luís, Marco Ângelo |
dc.subject.por.fl_str_mv |
Agmatina Dna Plasmídico Isoforma Superenrolada Suporte de Resina Terapêuticos de Dna Domínio/Área Científica::Engenharia e Tecnologia::Biotecnologia |
topic |
Agmatina Dna Plasmídico Isoforma Superenrolada Suporte de Resina Terapêuticos de Dna Domínio/Área Científica::Engenharia e Tecnologia::Biotecnologia |
description |
DNA-based therapeutics are one of the main applications aiming to find a course of treatment for hereditary diseases as well as cancer. They provide additional benefits when compared to traditional therapeutics. Plasmid DNA (pDNA) is one of the most utilized species of DNA-based therapeutics due to its ability to induce significant expression of a trans-gene and a comparably easy manufacturing process, without any of the disadvantages viral vectors show. This form of DNA presents linear, open circle and supercoiled isoforms, while being possible to switch between them, based on the amount and location of nicks in the sequence. The supercoiled isoform is the most biologically significant one since it allows for higher efficiencies of trans-gene expression. In order to use pDNA for human treatment, it needs to be purified so that the final product consists of a homogeneous supercoiled pDNA pool. To obtain this, a chromatography process with a combination of several interactions presents the best course of action, which is demonstrated by multiple products currently on the market. Nevertheless, the existing supports that are focused on the chromatographic step specific for the isolation of the relevant pDNA isoform (polishing step) do not present the best performance characteristics, both in terms of the purity obtained and the yield. Therefore, the present work aims to develop a novel polishing chromatographic material to produce supercoiled pDNA with higher purity and recovery. To do this, firstly, static binding experiments were conducted to confirm the results previously obtained by another working collaboration. Then, the promising prototypes were subjected to dynamic binding conditions, with the objective of assessing their capabilities to isolate the supercoiled isoform. This was followed by the development of several new agmatine-based prototypes where elution buffer composition and pH were optimized to achieve the highest resolution and purity of supercoiled pDNA. Afterwards, a step-elution method and optimization were conducted with a 3rd generation agmatine prototype, since it presented the best results in terms of supercoiled purity. With the optimized method, a complex sample of RNA and pDNA was applied to the prototype to evaluate if it was capable of separating supercoiled pDNA from other isoforms and similar molecules. The results indicated that the agmatine prototype is efficient in the purification of supercoiled pDNA from complex samples, with a high degree of purification, and in accordance with the specifications of regulatory agencies. |
publishDate |
2023 |
dc.date.none.fl_str_mv |
2023-07-18 2023-06-12 2023-07-18T00:00:00Z 9999-01-01T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10400.6/13622 TID:203381882 |
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eng |
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eng |
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metadata only access |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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