Accelerating Complex Biopharmaceuticals Manufactoring
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/10362/160882 |
Resumo: | Extracellular vesicles (EV) are phospholipid involucrum of cytosol, secreted by cells, implicated in inter-cellular communication, and enacting important roles in physiological and pathological processes. It is speculated they should give rise to a new class of gene therapy products, with clinical trials already under way. Viral vectors, on the other hand, are already used in gene and oncolytic therapy, as well as, for vaccines. Yet, the manufacturing process of such biopharmaceuticals is challenged by their complexity and heterogeneity. For EV as for lentiviral vectors (LV) the establishment of scalable and compatible with good manufacturing practices (cGMP) downstream processes (DSP) is still ongoing. The similarity between EV’s and LV’s chemical/physical properties inspired, in the present work, the devise of an affinity chromatography (AC) technology with two applications: one aimed at capturing EV; the other focused on the removal of product related impurities from LV feedstocks. Concomitantly, analytical tools based on fluorescence NTA (F-NTA) and biolayer interferometry (BLI) were developed. Using a CD81 ligand, intermediate scale experiments demonstrated the enrichment in EV, for which ~30% recoveries were obtained using vesicles from HEK293 and hiPSC. F-NTA proved to be unsuited for estimating the ratio of EV per total particles (TP), yet BLI showed potential for phenotype specific EV quantification, with <30% deviation from Interferometric Reflectance Imaging Sensor (SP-IRIS) results. Regarding LV purification, in a small-scale screening study, a 4-fold reduction in the ration between TP and transduction units (TU) was observed with CD81 and CD63 ligands. Furthermore, decrease in the TP/TU ratio was observed when utilizing purified material as load for AC, showcasing it could be added as a polishing step to current DSP processes. Altogether, this thesis contributed to the development of specific and efficient purification technologies which could potentiate the advent of EV-based gene therapies and/or complement current LV manufacturing pipelines. |
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Accelerating Complex Biopharmaceuticals Manufactoringaffinity chromatographybiolayer interferometrybiopharmaceuticalsdownstream processingextracellular vesicleslentiviral vectorsDomínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e TecnologiasExtracellular vesicles (EV) are phospholipid involucrum of cytosol, secreted by cells, implicated in inter-cellular communication, and enacting important roles in physiological and pathological processes. It is speculated they should give rise to a new class of gene therapy products, with clinical trials already under way. Viral vectors, on the other hand, are already used in gene and oncolytic therapy, as well as, for vaccines. Yet, the manufacturing process of such biopharmaceuticals is challenged by their complexity and heterogeneity. For EV as for lentiviral vectors (LV) the establishment of scalable and compatible with good manufacturing practices (cGMP) downstream processes (DSP) is still ongoing. The similarity between EV’s and LV’s chemical/physical properties inspired, in the present work, the devise of an affinity chromatography (AC) technology with two applications: one aimed at capturing EV; the other focused on the removal of product related impurities from LV feedstocks. Concomitantly, analytical tools based on fluorescence NTA (F-NTA) and biolayer interferometry (BLI) were developed. Using a CD81 ligand, intermediate scale experiments demonstrated the enrichment in EV, for which ~30% recoveries were obtained using vesicles from HEK293 and hiPSC. F-NTA proved to be unsuited for estimating the ratio of EV per total particles (TP), yet BLI showed potential for phenotype specific EV quantification, with <30% deviation from Interferometric Reflectance Imaging Sensor (SP-IRIS) results. Regarding LV purification, in a small-scale screening study, a 4-fold reduction in the ration between TP and transduction units (TU) was observed with CD81 and CD63 ligands. Furthermore, decrease in the TP/TU ratio was observed when utilizing purified material as load for AC, showcasing it could be added as a polishing step to current DSP processes. Altogether, this thesis contributed to the development of specific and efficient purification technologies which could potentiate the advent of EV-based gene therapies and/or complement current LV manufacturing pipelines.As vesículas extracelulares (VE) são nanopartículas fosfolipídicas secretadas pelas células, implicadas na comunicação intercelular. Dado o seu envolvimento em processos fisiológicos e patológicos, especula-se que venham a dar lugar a uma nova classe de terapias genéticas. Os vetores virais, por outro lado, são já utilizados em terapias génicas e oncolíticas, assim como em vacinas. Contudo, o processo de fabrico destes biofármacos é desafiado pela sua complexidade e heterogeneidade. Tanto para VE como para vetores lentivirais (VL) o estabelecimento de processos de purificação escaláveis encontra-se ainda em curso. A semelhança de propriedades físico-químicas entre ambos inspirou, no presente trabalho, a conceção de uma cromatografia de afinidade (CA) com duas aplicações: purificar VE; remover impurezas e VE dos meios de cultura de VL. Concomitantemente, ferramentas analíticas baseadas em NTA fluorescente (NTA-F) e interferometria de biocamada (IBC) foram desenvolvidas. Utilizando um ligando CD81, experiências em escala intermédia demonstraram enriquecimento em VE, nas quais rendimentos de ~30% foram obtidos para vesiculas de HEK293 e hiPSC. O NTA-F mostrou-se inadequado para estimar o rácio VE/partículas totais (PT), contudo a IBC demonstrou potencial na quantificação de vesículas com fenótipos específicos, tendo sido obtidos desvios <30% entre os títulos estimados e os obtidos por SP-IRIS. Relativamente à purificação de VL, num estudo de rastreio em pequena escala, uma redução em 4 vezes do rácio PT/unidades de transdução (UT) foi observado com os ligandos CD81 e CD63. Além disso, observou-se também um decréscimo no rácio PT/UT ao carregar material purificado em colunas anti-VE, demonstrando que esta tecnologia poderia ser adicionada como uma etapa de polimento aos atuais processos de purificação. Em suma, esta tese contribuiu para o desenvolvimento de tecnologias de purificação dotadas de especificidade e eficiência que poderão potenciar o advento de terapias genéticas à base de VE e/ou complementar as atuais cadeias de fabrico de VL.Peixoto, CristinaRUNRuiz, Afonso de Brito2023-11-222026-09-30T00:00:00Z2023-11-22T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/160882enginfo:eu-repo/semantics/embargoedAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T05:43:39Zoai:run.unl.pt:10362/160882Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:58:15.557090Repositório 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 |
Accelerating Complex Biopharmaceuticals Manufactoring |
title |
Accelerating Complex Biopharmaceuticals Manufactoring |
spellingShingle |
Accelerating Complex Biopharmaceuticals Manufactoring Ruiz, Afonso de Brito affinity chromatography biolayer interferometry biopharmaceuticals downstream processing extracellular vesicles lentiviral vectors Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
title_short |
Accelerating Complex Biopharmaceuticals Manufactoring |
title_full |
Accelerating Complex Biopharmaceuticals Manufactoring |
title_fullStr |
Accelerating Complex Biopharmaceuticals Manufactoring |
title_full_unstemmed |
Accelerating Complex Biopharmaceuticals Manufactoring |
title_sort |
Accelerating Complex Biopharmaceuticals Manufactoring |
author |
Ruiz, Afonso de Brito |
author_facet |
Ruiz, Afonso de Brito |
author_role |
author |
dc.contributor.none.fl_str_mv |
Peixoto, Cristina RUN |
dc.contributor.author.fl_str_mv |
Ruiz, Afonso de Brito |
dc.subject.por.fl_str_mv |
affinity chromatography biolayer interferometry biopharmaceuticals downstream processing extracellular vesicles lentiviral vectors Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
topic |
affinity chromatography biolayer interferometry biopharmaceuticals downstream processing extracellular vesicles lentiviral vectors Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
description |
Extracellular vesicles (EV) are phospholipid involucrum of cytosol, secreted by cells, implicated in inter-cellular communication, and enacting important roles in physiological and pathological processes. It is speculated they should give rise to a new class of gene therapy products, with clinical trials already under way. Viral vectors, on the other hand, are already used in gene and oncolytic therapy, as well as, for vaccines. Yet, the manufacturing process of such biopharmaceuticals is challenged by their complexity and heterogeneity. For EV as for lentiviral vectors (LV) the establishment of scalable and compatible with good manufacturing practices (cGMP) downstream processes (DSP) is still ongoing. The similarity between EV’s and LV’s chemical/physical properties inspired, in the present work, the devise of an affinity chromatography (AC) technology with two applications: one aimed at capturing EV; the other focused on the removal of product related impurities from LV feedstocks. Concomitantly, analytical tools based on fluorescence NTA (F-NTA) and biolayer interferometry (BLI) were developed. Using a CD81 ligand, intermediate scale experiments demonstrated the enrichment in EV, for which ~30% recoveries were obtained using vesicles from HEK293 and hiPSC. F-NTA proved to be unsuited for estimating the ratio of EV per total particles (TP), yet BLI showed potential for phenotype specific EV quantification, with <30% deviation from Interferometric Reflectance Imaging Sensor (SP-IRIS) results. Regarding LV purification, in a small-scale screening study, a 4-fold reduction in the ration between TP and transduction units (TU) was observed with CD81 and CD63 ligands. Furthermore, decrease in the TP/TU ratio was observed when utilizing purified material as load for AC, showcasing it could be added as a polishing step to current DSP processes. Altogether, this thesis contributed to the development of specific and efficient purification technologies which could potentiate the advent of EV-based gene therapies and/or complement current LV manufacturing pipelines. |
publishDate |
2023 |
dc.date.none.fl_str_mv |
2023-11-22 2023-11-22T00:00:00Z 2026-09-30T00:00:00Z |
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/10362/160882 |
url |
http://hdl.handle.net/10362/160882 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
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info:eu-repo/semantics/embargoedAccess |
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embargoedAccess |
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application/pdf |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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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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