Estudo de bioensaios e métodos cromatográficos para avaliação de potência/teor de toxina botulínica tipo A
Ano de defesa: | 2022 |
---|---|
Autor(a) principal: | |
Orientador(a): | |
Banca de defesa: | , , , |
Tipo de documento: | Tese |
Tipo de acesso: | Acesso aberto |
Idioma: | por |
Instituição de defesa: |
Universidade Federal de Santa Maria
Centro de Ciências da Saúde |
Programa de Pós-Graduação: |
Programa de Pós-Graduação em Ciências Farmacêuticas
|
Departamento: |
Farmácia
|
País: |
Brasil
|
Palavras-chave em Português: | |
Palavras-chave em Inglês: | |
Área do conhecimento CNPq: | |
Link de acesso: | http://repositorio.ufsm.br/handle/1/26603 |
Resumo: | Botulinum neurotoxin type A (BoNTA) belongs to a family of toxins which are produced by Clostridium botulinum. Their clinical use was approved in 1989 and since then, was used for therapeutic and aesthetic purposes. In the present study, a cell culture in vitro bioassay based on the cell line of adenocarcinoma T-47D, was studied and validated for the potency assessment of BoNTA in biopharmaceutical formulations. The validation studies showed that the bioassay is specific, accurate, repeatable and robust, and can be applied for the content/potency assessment of BoNTA. The results were compared with those of the LD50 bioassay pharmacopeial method, showing mean values 1.08% higher. The variance analysis (ANOVA) showed non-significant differences between the methods, at the level of significance of 5% (p = 0.05). The analysis of BoNTA in pharmaceutical products from different manufactures were analyzed by size exclusion and reversed-phase liquid chromatography, giving mean values 1.15% higher and 0.85% lower, respectively, compared to the T–47 cell culture bioassay. Besides, stability studies were carried out for evaluate the integrity of the biomolecule during the storage period of 36 months. The analysis showed decrease of the content of BoNTA, and allowed to quantify the higher molecular weight proteins and related proteins generated, that were lower than 3.63 and 1.10%, respectively. In this context, the study performed by physicochemical and biological methods represents an advance for the characterization, and evaluation of identity, purity, potency and stability of these biotechnology products. The methodologies can be applied for the production steps, purification and quality control, to ensure batch-to-batch consistency of BoNTA. Besides, represent important contribution in the context of the development of alternative methods to use of animals for the potency assessment of BoNTA, improving the quality control of biopharmaceutical formulations. |
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2022-10-19T22:08:57Z2022-10-19T22:08:57Z2022-07-19http://repositorio.ufsm.br/handle/1/26603Botulinum neurotoxin type A (BoNTA) belongs to a family of toxins which are produced by Clostridium botulinum. Their clinical use was approved in 1989 and since then, was used for therapeutic and aesthetic purposes. In the present study, a cell culture in vitro bioassay based on the cell line of adenocarcinoma T-47D, was studied and validated for the potency assessment of BoNTA in biopharmaceutical formulations. The validation studies showed that the bioassay is specific, accurate, repeatable and robust, and can be applied for the content/potency assessment of BoNTA. The results were compared with those of the LD50 bioassay pharmacopeial method, showing mean values 1.08% higher. The variance analysis (ANOVA) showed non-significant differences between the methods, at the level of significance of 5% (p = 0.05). The analysis of BoNTA in pharmaceutical products from different manufactures were analyzed by size exclusion and reversed-phase liquid chromatography, giving mean values 1.15% higher and 0.85% lower, respectively, compared to the T–47 cell culture bioassay. Besides, stability studies were carried out for evaluate the integrity of the biomolecule during the storage period of 36 months. The analysis showed decrease of the content of BoNTA, and allowed to quantify the higher molecular weight proteins and related proteins generated, that were lower than 3.63 and 1.10%, respectively. In this context, the study performed by physicochemical and biological methods represents an advance for the characterization, and evaluation of identity, purity, potency and stability of these biotechnology products. The methodologies can be applied for the production steps, purification and quality control, to ensure batch-to-batch consistency of BoNTA. Besides, represent important contribution in the context of the development of alternative methods to use of animals for the potency assessment of BoNTA, improving the quality control of biopharmaceutical formulations.A neurotoxina botulínica tipo A (BoNTA) pertence a uma família de toxinas produzidas pelo Clostridium botulinum. Seu uso clínico foi aprovado em 1989 e desde então, seus usos se expandiram para indicações terapêuticas e estéticas. No presente trabalho foi estudado e validado bioensaio por cultura de células in vitro para avaliação de potência de BoNTA em formulações biofarmacêuticas, utilizando a linhagem celular de adenocarcinoma T-47D. Os estudos de validação demonstraram que o bioensaio é específico, exato, robusto e reprodutível, e pode ser empregado para análise de teor/potência de BoNTA. Os resultados foram comparados aos fornecidos pelo método farmacopeico, o bioensaio da dose letal 50 in vivo, observando-se médias de potência 1,08% superiores. A análise de variância ANOVA demonstrou não haver diferenças estatisticamente significativas entre os dados fornecidos pelos métodos, ao nível de significância de 5% (p = 0,05). Realizou-se também, análise de BoNTA em produtos de diferentes empresas farmacêuticas por cromatografia líquida por exclusão molecular (CL–EM) e em fase reversa (CL–FR), obtendo-se diferença das médias dos valores de teor/potência 1,15% superior, e 0,85% inferior, respectivamente, em relação ao bioensaio por cultura de células T47D. Além disso, foram executados estudos de estabilidade para avaliar a integridade da molécula ao longo do período de armazenamento de 36 meses. Demonstrou-se a diminuição do teor de BoNTA e foram quantificadas as proteínas de alta massa molecular e proteínas relacionadas formadas, que foram inferiores a 3,63 e 1,10%, respectivamente. Neste contexto, os estudos realizados pelos métodos físico-químicos e biológicos constituem-se em avanços para a caracterização e avaliação de identidade, pureza, potência e estabilidade desses produtos biotecnológicos. As metodologias podem ser adotadas nas etapas de produção, purificação e controle de qualidade, garantindo a consistência lote-a-lote de BoNTA. Além disso, representam contribuição importante no contexto do desenvolvimento de métodos alternativos ao uso de animais para a avaliação de potência de BoNTA, aprimorando o controle da qualidade das formulações biofarmacêuticas.porUniversidade Federal de Santa MariaCentro de Ciências da SaúdePrograma de Pós-Graduação em Ciências FarmacêuticasUFSMBrasilFarmáciaAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessNeurotoxina botulínica tipo A (BoNTA)Bioensaio por cultura de células T–47DBioensaio in vivo da DL50Cromatografia líquida por exclusão molecular (CL–EM)Cromatografia líquida em fase reversa (CL–FR)Métodos alternativosBotulinum neurotoxin type A (BoNTA)T–47D cell culture bioassayLD50 in vivo bioassaySize exclusion liquid chromatography (SE–LC)Reversed phase liquid chromatography (RP–LC)Alternative methodsCNPQ::CIENCIAS DA SAUDE::FARMACIAEstudo de bioensaios e métodos cromatográficos para avaliação de potência/teor de toxina botulínica tipo AStudy of bioassays and chromatographic methods for the content/potency assessment of botulinum toxin type Ainfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisDalmora, Sergio Luizhttp://lattes.cnpq.br/4505166045049607Soares, Carlos Roberto JorgeMaldaner, Fernanda Pavani StammSouto, Ricardo BizogneMacedo, Rui Oliveirahttp://lattes.cnpq.br/1761737616973558Xavier, Bruna400300000005600600600600600600600c5c3d2e7-af0f-4f36-b006-046daf37340ad0872be6-de96-46bf-a050-30c79e8ae008988761d9-e899-4ae5-af15-730e68f1a4d4ac99a227-2675-48a9-96bf-6bc6ee6e8d5acf76b7c6-598e-40f0-9a07-08760c74cbd3bc2f55a2-e6a1-4986-a8b4-a737a89ff5bareponame:Manancial - Repositório Digital da UFSMinstname:Universidade Federal de Santa Maria (UFSM)instacron:UFSMCC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; 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dc.title.por.fl_str_mv |
Estudo de bioensaios e métodos cromatográficos para avaliação de potência/teor de toxina botulínica tipo A |
dc.title.alternative.eng.fl_str_mv |
Study of bioassays and chromatographic methods for the content/potency assessment of botulinum toxin type A |
title |
Estudo de bioensaios e métodos cromatográficos para avaliação de potência/teor de toxina botulínica tipo A |
spellingShingle |
Estudo de bioensaios e métodos cromatográficos para avaliação de potência/teor de toxina botulínica tipo A Xavier, Bruna Neurotoxina botulínica tipo A (BoNTA) Bioensaio por cultura de células T–47D Bioensaio in vivo da DL50 Cromatografia líquida por exclusão molecular (CL–EM) Cromatografia líquida em fase reversa (CL–FR) Métodos alternativos Botulinum neurotoxin type A (BoNTA) T–47D cell culture bioassay LD50 in vivo bioassay Size exclusion liquid chromatography (SE–LC) Reversed phase liquid chromatography (RP–LC) Alternative methods CNPQ::CIENCIAS DA SAUDE::FARMACIA |
title_short |
Estudo de bioensaios e métodos cromatográficos para avaliação de potência/teor de toxina botulínica tipo A |
title_full |
Estudo de bioensaios e métodos cromatográficos para avaliação de potência/teor de toxina botulínica tipo A |
title_fullStr |
Estudo de bioensaios e métodos cromatográficos para avaliação de potência/teor de toxina botulínica tipo A |
title_full_unstemmed |
Estudo de bioensaios e métodos cromatográficos para avaliação de potência/teor de toxina botulínica tipo A |
title_sort |
Estudo de bioensaios e métodos cromatográficos para avaliação de potência/teor de toxina botulínica tipo A |
author |
Xavier, Bruna |
author_facet |
Xavier, Bruna |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Dalmora, Sergio Luiz |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/4505166045049607 |
dc.contributor.referee1.fl_str_mv |
Soares, Carlos Roberto Jorge |
dc.contributor.referee2.fl_str_mv |
Maldaner, Fernanda Pavani Stamm |
dc.contributor.referee3.fl_str_mv |
Souto, Ricardo Bizogne |
dc.contributor.referee4.fl_str_mv |
Macedo, Rui Oliveira |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/1761737616973558 |
dc.contributor.author.fl_str_mv |
Xavier, Bruna |
contributor_str_mv |
Dalmora, Sergio Luiz Soares, Carlos Roberto Jorge Maldaner, Fernanda Pavani Stamm Souto, Ricardo Bizogne Macedo, Rui Oliveira |
dc.subject.por.fl_str_mv |
Neurotoxina botulínica tipo A (BoNTA) Bioensaio por cultura de células T–47D Bioensaio in vivo da DL50 Cromatografia líquida por exclusão molecular (CL–EM) Cromatografia líquida em fase reversa (CL–FR) Métodos alternativos |
topic |
Neurotoxina botulínica tipo A (BoNTA) Bioensaio por cultura de células T–47D Bioensaio in vivo da DL50 Cromatografia líquida por exclusão molecular (CL–EM) Cromatografia líquida em fase reversa (CL–FR) Métodos alternativos Botulinum neurotoxin type A (BoNTA) T–47D cell culture bioassay LD50 in vivo bioassay Size exclusion liquid chromatography (SE–LC) Reversed phase liquid chromatography (RP–LC) Alternative methods CNPQ::CIENCIAS DA SAUDE::FARMACIA |
dc.subject.eng.fl_str_mv |
Botulinum neurotoxin type A (BoNTA) T–47D cell culture bioassay LD50 in vivo bioassay Size exclusion liquid chromatography (SE–LC) Reversed phase liquid chromatography (RP–LC) Alternative methods |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS DA SAUDE::FARMACIA |
description |
Botulinum neurotoxin type A (BoNTA) belongs to a family of toxins which are produced by Clostridium botulinum. Their clinical use was approved in 1989 and since then, was used for therapeutic and aesthetic purposes. In the present study, a cell culture in vitro bioassay based on the cell line of adenocarcinoma T-47D, was studied and validated for the potency assessment of BoNTA in biopharmaceutical formulations. The validation studies showed that the bioassay is specific, accurate, repeatable and robust, and can be applied for the content/potency assessment of BoNTA. The results were compared with those of the LD50 bioassay pharmacopeial method, showing mean values 1.08% higher. The variance analysis (ANOVA) showed non-significant differences between the methods, at the level of significance of 5% (p = 0.05). The analysis of BoNTA in pharmaceutical products from different manufactures were analyzed by size exclusion and reversed-phase liquid chromatography, giving mean values 1.15% higher and 0.85% lower, respectively, compared to the T–47 cell culture bioassay. Besides, stability studies were carried out for evaluate the integrity of the biomolecule during the storage period of 36 months. The analysis showed decrease of the content of BoNTA, and allowed to quantify the higher molecular weight proteins and related proteins generated, that were lower than 3.63 and 1.10%, respectively. In this context, the study performed by physicochemical and biological methods represents an advance for the characterization, and evaluation of identity, purity, potency and stability of these biotechnology products. The methodologies can be applied for the production steps, purification and quality control, to ensure batch-to-batch consistency of BoNTA. Besides, represent important contribution in the context of the development of alternative methods to use of animals for the potency assessment of BoNTA, improving the quality control of biopharmaceutical formulations. |
publishDate |
2022 |
dc.date.accessioned.fl_str_mv |
2022-10-19T22:08:57Z |
dc.date.available.fl_str_mv |
2022-10-19T22:08:57Z |
dc.date.issued.fl_str_mv |
2022-07-19 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
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doctoralThesis |
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http://repositorio.ufsm.br/handle/1/26603 |
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http://repositorio.ufsm.br/handle/1/26603 |
dc.language.iso.fl_str_mv |
por |
language |
por |
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400300000005 |
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600 600 600 600 600 600 600 |
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Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ info:eu-repo/semantics/openAccess |
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Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ |
eu_rights_str_mv |
openAccess |
dc.publisher.none.fl_str_mv |
Universidade Federal de Santa Maria Centro de Ciências da Saúde |
dc.publisher.program.fl_str_mv |
Programa de Pós-Graduação em Ciências Farmacêuticas |
dc.publisher.initials.fl_str_mv |
UFSM |
dc.publisher.country.fl_str_mv |
Brasil |
dc.publisher.department.fl_str_mv |
Farmácia |
publisher.none.fl_str_mv |
Universidade Federal de Santa Maria Centro de Ciências da Saúde |
dc.source.none.fl_str_mv |
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