Recombinant human interferon analysis in pharmaceutical formulations
Autor(a) principal: | |
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Data de Publicação: | 2017 |
Outros Autores: | , |
Tipo de documento: | Artigo |
Idioma: | por eng |
Título da fonte: | Vigilância Sanitária em Debate |
Texto Completo: | https://visaemdebate.incqs.fiocruz.br/index.php/visaemdebate/article/view/943 |
Resumo: | Introduction: Due to the interest in the treatment of hepatitis, the industrial production process of INF-α has been developed and perfected over the last few years. Objective: The present work aimed to develop a protocol to characterize the molecular structure of INF-α2b in pharmaceutical formulations by MALDI-TOF mass spectrometry. Method: Initially, a reversed-phase liquid chromatography method was developed to promote the separation of active and minor constituent INF-α2b and human serum albumin, also present in the pharmaceutical formulations, to obtain samples with protein homogeneity revealed by electrophoresis. Samples were hydrolyzed with trypsin and submitted to MALDI-TOF. In order to analyze the molecular structure, a procedure based on immunoaffinity and gel filtration chromatography was developed. Results: Prepared samples by these methods showed protein homogeneity by SDS-PAGE, and were analyzed by circular dichroism and fluorescence, which showed three – dimensional structure degradation. Conclusions: This work provides important data that support the establishment of a protocol for the analysis of INF-α2b in final product, which could replace the traditional peptide mapping by liquid chromatography, with the advantage of resulting in a larger amount of information about the structure of the biopharmaceutical. |
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Recombinant human interferon analysis in pharmaceutical formulationsAnálise de interferon humano recombinante presente em formulações farmacêuticasInterferon-alfaProteínasMALDI-TOFDicroísmo CircularFluorescênciaAlpha InterferonProteinMALDI-TOFCircular DichroismFluorescenceIntroduction: Due to the interest in the treatment of hepatitis, the industrial production process of INF-α has been developed and perfected over the last few years. Objective: The present work aimed to develop a protocol to characterize the molecular structure of INF-α2b in pharmaceutical formulations by MALDI-TOF mass spectrometry. Method: Initially, a reversed-phase liquid chromatography method was developed to promote the separation of active and minor constituent INF-α2b and human serum albumin, also present in the pharmaceutical formulations, to obtain samples with protein homogeneity revealed by electrophoresis. Samples were hydrolyzed with trypsin and submitted to MALDI-TOF. In order to analyze the molecular structure, a procedure based on immunoaffinity and gel filtration chromatography was developed. Results: Prepared samples by these methods showed protein homogeneity by SDS-PAGE, and were analyzed by circular dichroism and fluorescence, which showed three – dimensional structure degradation. Conclusions: This work provides important data that support the establishment of a protocol for the analysis of INF-α2b in final product, which could replace the traditional peptide mapping by liquid chromatography, with the advantage of resulting in a larger amount of information about the structure of the biopharmaceutical.Introdução: Em virtude do interesse para o tratamento da hepatite, o processo de produção industrial do INF-α foi desenvolvido e aperfeiçoado ao longo dos últimos anos. Objetivo: O presente trabalho teve como objetivo desenvolver um protocolo para caracterizar a estrutura molecular do INF-α2b em formulações farmacêuticas por espectrometria de massa do tipo MALDI-TOF. Método: Inicialmente foi desenvolvido um método de cromatografia líquida baseado em fase reversa para promover a separação entre o INF-α2b constituinte ativo e minoritário e a soro albumina humana, também componente presente nas formulações farmacêuticas, obtendo-se amostras com homogeneidade proteica, revelada por eletroforese. As amostras em solução foram submetidas à digestão com tripsina, levadas ao espectrômetro de massa MALDI-TOF. Para que fosse analisada a estrutura molecular, foi desenvolvido um procedimento baseado em imunoafinidade e cromatografia de gel filtração. Resultados: As amostras preparadas por estes métodos apresentaram homogeneidade proteica por eletroforese, sendo analisadas por dicroísmo circular e fluorescência, o que demonstrou ter degradação da estrutura tridimensional. Conclusões: Esse trabalho fornece dados importantes que subsidiam o estabelecimento de um protocolo para a análise de INF-α2b em produto final, que poderia substituir o mapa de peptídeos tradicional por cromatografia líquida, com a vantagem de resultar em um maior número de informações sobre a estrutura molecular do biofármaco.Instituto Nacional de Controle de Qualidade em Saúde2017-08-31info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersion"Editor de seção"application/pdfapplication/pdfhttps://visaemdebate.incqs.fiocruz.br/index.php/visaemdebate/article/view/94310.22239/2317-269X.00943Health Surveillance under Debate: Society, Science & Technology ; Vol. 5 No. 3 (2017): August; 66-75Vigilancia en Salud en Debate: Sociedad, Ciencia y Tecnología; Vol. 5 Núm. 3 (2017): Agosto; 66-75Vigil Sanit Debate, Rio de Janeiro; v. 5 n. 3 (2017): Agosto; 66-752317-269Xreponame:Vigilância Sanitária em Debateinstname:Fundação Oswaldo Cruz (Fiocruz)instacron:FIOCRUZporenghttps://visaemdebate.incqs.fiocruz.br/index.php/visaemdebate/article/view/943/402https://visaemdebate.incqs.fiocruz.br/index.php/visaemdebate/article/view/943/566Copyright (c) 2017 Vigilância Sanitária em Debate: Sociedade, Ciência & Tecnologia (Health Surveillance under Debate: Society, Science & Technology) – Visa em Debatehttps://creativecommons.org/licenses/by-nc-nd/4.0info:eu-repo/semantics/openAccessde Andrade, Sinéa Mendesda Silva, ManuelaSilva, Filipe Soares Quirino da2021-09-30T19:22:19Zoai:ojs.visaemdebate.incqs.fiocruz.br:article/943Revistahttps://visaemdebate.incqs.fiocruz.br/index.php/visaemdebatePUBhttps://visaemdebate.incqs.fiocruz.br/index.php/visaemdebate/oaiincqs.visaemdebate@fiocruz.br || gisele.neves@fiocruz.br2317-269X2317-269Xopendoar:2021-09-30T19:22:19Vigilância Sanitária em Debate - Fundação Oswaldo Cruz (Fiocruz)false |
dc.title.none.fl_str_mv |
Recombinant human interferon analysis in pharmaceutical formulations Análise de interferon humano recombinante presente em formulações farmacêuticas |
title |
Recombinant human interferon analysis in pharmaceutical formulations |
spellingShingle |
Recombinant human interferon analysis in pharmaceutical formulations de Andrade, Sinéa Mendes Interferon-alfa Proteínas MALDI-TOF Dicroísmo Circular Fluorescência Alpha Interferon Protein MALDI-TOF Circular Dichroism Fluorescence |
title_short |
Recombinant human interferon analysis in pharmaceutical formulations |
title_full |
Recombinant human interferon analysis in pharmaceutical formulations |
title_fullStr |
Recombinant human interferon analysis in pharmaceutical formulations |
title_full_unstemmed |
Recombinant human interferon analysis in pharmaceutical formulations |
title_sort |
Recombinant human interferon analysis in pharmaceutical formulations |
author |
de Andrade, Sinéa Mendes |
author_facet |
de Andrade, Sinéa Mendes da Silva, Manuela Silva, Filipe Soares Quirino da |
author_role |
author |
author2 |
da Silva, Manuela Silva, Filipe Soares Quirino da |
author2_role |
author author |
dc.contributor.author.fl_str_mv |
de Andrade, Sinéa Mendes da Silva, Manuela Silva, Filipe Soares Quirino da |
dc.subject.por.fl_str_mv |
Interferon-alfa Proteínas MALDI-TOF Dicroísmo Circular Fluorescência Alpha Interferon Protein MALDI-TOF Circular Dichroism Fluorescence |
topic |
Interferon-alfa Proteínas MALDI-TOF Dicroísmo Circular Fluorescência Alpha Interferon Protein MALDI-TOF Circular Dichroism Fluorescence |
description |
Introduction: Due to the interest in the treatment of hepatitis, the industrial production process of INF-α has been developed and perfected over the last few years. Objective: The present work aimed to develop a protocol to characterize the molecular structure of INF-α2b in pharmaceutical formulations by MALDI-TOF mass spectrometry. Method: Initially, a reversed-phase liquid chromatography method was developed to promote the separation of active and minor constituent INF-α2b and human serum albumin, also present in the pharmaceutical formulations, to obtain samples with protein homogeneity revealed by electrophoresis. Samples were hydrolyzed with trypsin and submitted to MALDI-TOF. In order to analyze the molecular structure, a procedure based on immunoaffinity and gel filtration chromatography was developed. Results: Prepared samples by these methods showed protein homogeneity by SDS-PAGE, and were analyzed by circular dichroism and fluorescence, which showed three – dimensional structure degradation. Conclusions: This work provides important data that support the establishment of a protocol for the analysis of INF-α2b in final product, which could replace the traditional peptide mapping by liquid chromatography, with the advantage of resulting in a larger amount of information about the structure of the biopharmaceutical. |
publishDate |
2017 |
dc.date.none.fl_str_mv |
2017-08-31 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion "Editor de seção" |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://visaemdebate.incqs.fiocruz.br/index.php/visaemdebate/article/view/943 10.22239/2317-269X.00943 |
url |
https://visaemdebate.incqs.fiocruz.br/index.php/visaemdebate/article/view/943 |
identifier_str_mv |
10.22239/2317-269X.00943 |
dc.language.iso.fl_str_mv |
por eng |
language |
por eng |
dc.relation.none.fl_str_mv |
https://visaemdebate.incqs.fiocruz.br/index.php/visaemdebate/article/view/943/402 https://visaemdebate.incqs.fiocruz.br/index.php/visaemdebate/article/view/943/566 |
dc.rights.driver.fl_str_mv |
https://creativecommons.org/licenses/by-nc-nd/4.0 info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
https://creativecommons.org/licenses/by-nc-nd/4.0 |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf application/pdf |
dc.publisher.none.fl_str_mv |
Instituto Nacional de Controle de Qualidade em Saúde |
publisher.none.fl_str_mv |
Instituto Nacional de Controle de Qualidade em Saúde |
dc.source.none.fl_str_mv |
Health Surveillance under Debate: Society, Science & Technology ; Vol. 5 No. 3 (2017): August; 66-75 Vigilancia en Salud en Debate: Sociedad, Ciencia y Tecnología; Vol. 5 Núm. 3 (2017): Agosto; 66-75 Vigil Sanit Debate, Rio de Janeiro; v. 5 n. 3 (2017): Agosto; 66-75 2317-269X reponame:Vigilância Sanitária em Debate instname:Fundação Oswaldo Cruz (Fiocruz) instacron:FIOCRUZ |
instname_str |
Fundação Oswaldo Cruz (Fiocruz) |
instacron_str |
FIOCRUZ |
institution |
FIOCRUZ |
reponame_str |
Vigilância Sanitária em Debate |
collection |
Vigilância Sanitária em Debate |
repository.name.fl_str_mv |
Vigilância Sanitária em Debate - Fundação Oswaldo Cruz (Fiocruz) |
repository.mail.fl_str_mv |
incqs.visaemdebate@fiocruz.br || gisele.neves@fiocruz.br |
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1797042047434096640 |