Extraction and purification of immunoglobulin G with aqueous biphasic systems
Autor(a) principal: | |
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Data de Publicação: | 2015 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10773/16144 |
Resumo: | The immune system is able to produce antibodies, which have the capacity to recognize and to bind to foreign molecules or pathogenic organisms. Currently, there are a diversity of diseases that can be treated with antibodies, like immunoglobulins G (IgG). Thereby, the development of cost-efficient processes for their extraction and purification is an area of main interest in biotechnology. Aqueous biphasic systems (ABS) have been investigated for this purpose, once they allow the reduction of costs and the number of steps involved in the process, when compared with conventional methods. Nevertheless, typical ABS have not showed to be selective, resulting in low purification factors and yields. In this context, the addition of ionic liquids (ILs) as adjuvants can be a viable and potential alternative to tailor the selectivity of these systems. In this work, ABS composed of polyethylene glycol (PEG) of different molecular weight, and a biodegradable salt (potassium citrate) using ILs as adjuvants (5 wt%), were studied for the extraction and purification of IgG from a rabbit source. Initially, it was tested the extraction time, the effect on the molecular weight of PEG in a buffer solution of K3C6H5O7/C6H8O7 at pH≈7, and the effect of pH (59) on the yield (YIgG) and extraction efficiency (EEIgG%) of IgG. The best results regarding EEIgG% were achieved with a centrifugation step at 1000 rpm, during 10 min, in order to promote the separation of phases followed by 120 min of equilibrium. This procedure was then applied to the remaining experiments. The results obtained in the study of PEGs with different molecular weights, revealed a high affinity of IgG for the PEG-rich phase, and particularly for PEGs of lower molecular weight (EEIgG% of 96 % with PEG 400). On the other hand, the variation of pH in the buffer solution did not show a significant effect on the EEIgG%. Finally, it was evaluated the influence of the addition of different ILs (5% wt) on the IgG extraction in ABS composed of PEG 400 at pH≈7. In these studies, it was possible to obtain EEIgG% of 100% with the ILs composed of the anions [TOS]-, [CH3CO2]-and Cl-, although the obtained YIgG% were lower than 40%. On the other hand, the ILs composed of the anions Br-, as well as of the cation [C10mim]+, although not leading to EEIgG% of 100%, provide an increase in the YIgG%. ABS composed of PEG, a biodegradable organic salt and ILs as adjuvants, revealed to be an alternative and promising method to purify IgG. However, additional studies are still required in order to reduce the loss of IgG. |
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Extraction and purification of immunoglobulin G with aqueous biphasic systemsBiotecnologia industrialSistemas aquosos bifásicosImunoglobulinasExtracção líquido-líquidoSoluções iónicasThe immune system is able to produce antibodies, which have the capacity to recognize and to bind to foreign molecules or pathogenic organisms. Currently, there are a diversity of diseases that can be treated with antibodies, like immunoglobulins G (IgG). Thereby, the development of cost-efficient processes for their extraction and purification is an area of main interest in biotechnology. Aqueous biphasic systems (ABS) have been investigated for this purpose, once they allow the reduction of costs and the number of steps involved in the process, when compared with conventional methods. Nevertheless, typical ABS have not showed to be selective, resulting in low purification factors and yields. In this context, the addition of ionic liquids (ILs) as adjuvants can be a viable and potential alternative to tailor the selectivity of these systems. In this work, ABS composed of polyethylene glycol (PEG) of different molecular weight, and a biodegradable salt (potassium citrate) using ILs as adjuvants (5 wt%), were studied for the extraction and purification of IgG from a rabbit source. Initially, it was tested the extraction time, the effect on the molecular weight of PEG in a buffer solution of K3C6H5O7/C6H8O7 at pH≈7, and the effect of pH (59) on the yield (YIgG) and extraction efficiency (EEIgG%) of IgG. The best results regarding EEIgG% were achieved with a centrifugation step at 1000 rpm, during 10 min, in order to promote the separation of phases followed by 120 min of equilibrium. This procedure was then applied to the remaining experiments. The results obtained in the study of PEGs with different molecular weights, revealed a high affinity of IgG for the PEG-rich phase, and particularly for PEGs of lower molecular weight (EEIgG% of 96 % with PEG 400). On the other hand, the variation of pH in the buffer solution did not show a significant effect on the EEIgG%. Finally, it was evaluated the influence of the addition of different ILs (5% wt) on the IgG extraction in ABS composed of PEG 400 at pH≈7. In these studies, it was possible to obtain EEIgG% of 100% with the ILs composed of the anions [TOS]-, [CH3CO2]-and Cl-, although the obtained YIgG% were lower than 40%. On the other hand, the ILs composed of the anions Br-, as well as of the cation [C10mim]+, although not leading to EEIgG% of 100%, provide an increase in the YIgG%. ABS composed of PEG, a biodegradable organic salt and ILs as adjuvants, revealed to be an alternative and promising method to purify IgG. However, additional studies are still required in order to reduce the loss of IgG.O sistema imunitário tem como ferramenta mais poderosa a produção de anticorpos, pois estes têm a capacidade de reconhecer e ligar-se a moléculas/organismos patogénicos. Atualmente, existem uma série de doenças que podem ser tratadas com anticorpos, nomeadamente com as imunoglobulinas G (IgG). Assim, o desenvolvimento de processos mais baratos e eficazes para a sua extração e purificação é uma área de interesse em biotecnologia. Os sistemas aquosos bifásicos (SAB) têm sido estudados para o efeito pois permitem a redução de custos e do número de passos envolvidos no processo, em comparação com os métodos convencionais. No entanto, os SAB tradicionais têm-se mostrado pouco seletivos, resultando em baixos fatores de purificação e rendimentos. Neste sentido, a incorporação de líquidos iónicos (LIs) nos SAB pode ser uma alternativa promissora para manipular a seletividade destes sistemas. Neste trabalho, estudaram-se SAB constituídos por polietileno glicol (PEG) de diferentes massas moleculares e um sal biodegradável (citrato de potássio), utilizando LIs como adjuvantes (5% m/m) para a extração de IgG de origem leporídea (coelho). Inicialmente, estudaram-se o tempo de extração, o efeito da variação da massa molecular do PEG em solução tampão de K3C6H5O7/C6H8O7 a pH≈7 e o efeito da variação de pH (5-9) sobre o rendimento (YIgG) e eficiência de extração (EEIgG%) de IgG. Os melhores resultados obtidos em termos de EEIgG% foram conseguidos com uma centrifugação a 1000 rpm durante 10 min para promover a separação de fases e equílibrio seguidos de 120 min de repouso. Este procedimento foi posteriormente aplicado aos restantes ensaios experimentais. Os resultados obtidos no estudo de PEGs de diferentes pesos moleculares revelaram uma elevada afinidade da IgG para a fase de PEG, principalmente com os PEGs de menor peso molecular (96% de EEIgG% com o PEG 400). Por outro lado, a variação de pH da solução tampão não mostrou um efeito significativo sobre a EEIgG%. Por fim, foi avaliada a influência da adição de vários LIs (5% m/m) na extração de IgG nos SABs compostos por PEG 400 a pH≈7. Nestes estudos foi possível obter uma EEIgG% de 100% com os LIs constituídos pelos aniões [TOS]-, [CH3CO2]- e Cl-, apesar dos YIgG% obtidos serem inferiores a 40%. Por outro lado, com LIs constituídos pelos aniões Br-, assim como pelos catião [C10mim]+, não se conseguiram obter valores de 100% para a EEIgG%, mas obtiveram-se melhores resultados em termos de YIgG%. Os SAB constituídos por PEG, um sal orgânico biodegradável e LIs como adjuvantes revelaram-se um método alternativo e promissor para a purificação de IgG. No entanto, são necessários ainda estudos adicionais de modo a reduzir a perda de IgG durante o processo.Universidade de Aveiro2018-07-20T14:00:56Z2015-07-31T00:00:00Z2015-07-312017-07-24T17:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10773/16144TID:201596806engFaustino, Vânia Filipa Martinsinfo: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:RCAAP2024-02-22T11:29:59Zoai:ria.ua.pt:10773/16144Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T02:51:19.801328Repositório 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 |
Extraction and purification of immunoglobulin G with aqueous biphasic systems |
title |
Extraction and purification of immunoglobulin G with aqueous biphasic systems |
spellingShingle |
Extraction and purification of immunoglobulin G with aqueous biphasic systems Faustino, Vânia Filipa Martins Biotecnologia industrial Sistemas aquosos bifásicos Imunoglobulinas Extracção líquido-líquido Soluções iónicas |
title_short |
Extraction and purification of immunoglobulin G with aqueous biphasic systems |
title_full |
Extraction and purification of immunoglobulin G with aqueous biphasic systems |
title_fullStr |
Extraction and purification of immunoglobulin G with aqueous biphasic systems |
title_full_unstemmed |
Extraction and purification of immunoglobulin G with aqueous biphasic systems |
title_sort |
Extraction and purification of immunoglobulin G with aqueous biphasic systems |
author |
Faustino, Vânia Filipa Martins |
author_facet |
Faustino, Vânia Filipa Martins |
author_role |
author |
dc.contributor.author.fl_str_mv |
Faustino, Vânia Filipa Martins |
dc.subject.por.fl_str_mv |
Biotecnologia industrial Sistemas aquosos bifásicos Imunoglobulinas Extracção líquido-líquido Soluções iónicas |
topic |
Biotecnologia industrial Sistemas aquosos bifásicos Imunoglobulinas Extracção líquido-líquido Soluções iónicas |
description |
The immune system is able to produce antibodies, which have the capacity to recognize and to bind to foreign molecules or pathogenic organisms. Currently, there are a diversity of diseases that can be treated with antibodies, like immunoglobulins G (IgG). Thereby, the development of cost-efficient processes for their extraction and purification is an area of main interest in biotechnology. Aqueous biphasic systems (ABS) have been investigated for this purpose, once they allow the reduction of costs and the number of steps involved in the process, when compared with conventional methods. Nevertheless, typical ABS have not showed to be selective, resulting in low purification factors and yields. In this context, the addition of ionic liquids (ILs) as adjuvants can be a viable and potential alternative to tailor the selectivity of these systems. In this work, ABS composed of polyethylene glycol (PEG) of different molecular weight, and a biodegradable salt (potassium citrate) using ILs as adjuvants (5 wt%), were studied for the extraction and purification of IgG from a rabbit source. Initially, it was tested the extraction time, the effect on the molecular weight of PEG in a buffer solution of K3C6H5O7/C6H8O7 at pH≈7, and the effect of pH (59) on the yield (YIgG) and extraction efficiency (EEIgG%) of IgG. The best results regarding EEIgG% were achieved with a centrifugation step at 1000 rpm, during 10 min, in order to promote the separation of phases followed by 120 min of equilibrium. This procedure was then applied to the remaining experiments. The results obtained in the study of PEGs with different molecular weights, revealed a high affinity of IgG for the PEG-rich phase, and particularly for PEGs of lower molecular weight (EEIgG% of 96 % with PEG 400). On the other hand, the variation of pH in the buffer solution did not show a significant effect on the EEIgG%. Finally, it was evaluated the influence of the addition of different ILs (5% wt) on the IgG extraction in ABS composed of PEG 400 at pH≈7. In these studies, it was possible to obtain EEIgG% of 100% with the ILs composed of the anions [TOS]-, [CH3CO2]-and Cl-, although the obtained YIgG% were lower than 40%. On the other hand, the ILs composed of the anions Br-, as well as of the cation [C10mim]+, although not leading to EEIgG% of 100%, provide an increase in the YIgG%. ABS composed of PEG, a biodegradable organic salt and ILs as adjuvants, revealed to be an alternative and promising method to purify IgG. However, additional studies are still required in order to reduce the loss of IgG. |
publishDate |
2015 |
dc.date.none.fl_str_mv |
2015-07-31T00:00:00Z 2015-07-31 2017-07-24T17:00:00Z 2018-07-20T14:00:56Z |
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/10773/16144 TID:201596806 |
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http://hdl.handle.net/10773/16144 |
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TID:201596806 |
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eng |
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eng |
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info:eu-repo/semantics/openAccess |
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openAccess |
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application/pdf |
dc.publisher.none.fl_str_mv |
Universidade de Aveiro |
publisher.none.fl_str_mv |
Universidade de Aveiro |
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reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
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RCAAP |
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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) |
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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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