Desenvolvimento e validação de métodos cromatográficos para avaliação de teor/potência de insulina glargina
Autor(a) principal: | |
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Data de Publicação: | 2016 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Manancial - Repositório Digital da UFSM |
Texto Completo: | http://repositorio.ufsm.br/handle/1/6037 |
Resumo: | hans, and is secreted into the bloodstream. It plays and important role in regulating the metabolic activities of the body, particularly the homeostasis of the blood glucose. Insulin glargine is a recombinant human insulin analogue produced by DNA technology using a strain of Escherichia coli and the insulin glargine differ only by three amino acids from human insulin. Reversed-phase liquid chromatography (RP-LC) and size exclusion liquid chromatography (SE-LC) methods were developed and validated for the assessment of insulin glargine in biopharmaceutical formulations. A RP-LC method was carried out on a Jupiter C4 column (250 mm x 4.6 mm i.d.), maintained at 30 ºC. The mobile phase A consisted of 0.05 M sodium sulphate buffer, pH 2.5, and the mobile phase B was acetonitrile. The SE-LC method was carried out on a BioSep-SEC-S 2000 column (300 mm x 7.8 mm i.d.), maintained at 25 ºC. The mobile phase consisted of 0.03 M MES acid buffer, pH 2.5, run isocratically at a flow rate of 0.6 mL/min. Chromatographic separation was obtained with retention times of 7.5 min, and 9.9 min, and was linear over the concentration range of 0.05 - 200 μg/mL (R2 = 0.9998) and 0.02 - 180 μg/mL (R2 = 0.9999), respectively, for RP-LC and SE-LC, with photodiode array (PDA) detection at 214 nm and 200 nm for RP-LC and SE-LC methods, respectively. The limits of detection and quantitation were 0.018 and 0.054 μg/mL, respectively, for the RP-LC and 0.009 and 0.027 μg/mL, for the SE-LC. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. Equally, the accuracy was 100.13% and 99.38%, with bias lower than 0.85% and than 0.86%. The validated methods were applied for the determination of insulin glargine and related proteins and high molecular mass, in biotechnology-derived products, giving lower mean differences of the estimated content/potencies of 0.21% and 0.16% for the RP-LC and SE-LC related, compared to the in vitro cell culture assay. It is concluded that represents a contribution to establish alternatives to monitor stability, quality control and thereby assure therapeutic efficacy of the biotechnology-derived medicine. |
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Desenvolvimento e validação de métodos cromatográficos para avaliação de teor/potência de insulina glarginaDevelopment and validation of chromatographic methods for the content/potency evaluation of insulin glargineInsulina glarginaCromatografia líquidaValidaçãoBioensaioCorrelaçãoInsulin glargineLiquid chromatography methodsValidationBioassayCorrelationCNPQ::CIENCIAS DA SAUDE::FARMACIAhans, and is secreted into the bloodstream. It plays and important role in regulating the metabolic activities of the body, particularly the homeostasis of the blood glucose. Insulin glargine is a recombinant human insulin analogue produced by DNA technology using a strain of Escherichia coli and the insulin glargine differ only by three amino acids from human insulin. Reversed-phase liquid chromatography (RP-LC) and size exclusion liquid chromatography (SE-LC) methods were developed and validated for the assessment of insulin glargine in biopharmaceutical formulations. A RP-LC method was carried out on a Jupiter C4 column (250 mm x 4.6 mm i.d.), maintained at 30 ºC. The mobile phase A consisted of 0.05 M sodium sulphate buffer, pH 2.5, and the mobile phase B was acetonitrile. The SE-LC method was carried out on a BioSep-SEC-S 2000 column (300 mm x 7.8 mm i.d.), maintained at 25 ºC. The mobile phase consisted of 0.03 M MES acid buffer, pH 2.5, run isocratically at a flow rate of 0.6 mL/min. Chromatographic separation was obtained with retention times of 7.5 min, and 9.9 min, and was linear over the concentration range of 0.05 - 200 μg/mL (R2 = 0.9998) and 0.02 - 180 μg/mL (R2 = 0.9999), respectively, for RP-LC and SE-LC, with photodiode array (PDA) detection at 214 nm and 200 nm for RP-LC and SE-LC methods, respectively. The limits of detection and quantitation were 0.018 and 0.054 μg/mL, respectively, for the RP-LC and 0.009 and 0.027 μg/mL, for the SE-LC. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. Equally, the accuracy was 100.13% and 99.38%, with bias lower than 0.85% and than 0.86%. The validated methods were applied for the determination of insulin glargine and related proteins and high molecular mass, in biotechnology-derived products, giving lower mean differences of the estimated content/potencies of 0.21% and 0.16% for the RP-LC and SE-LC related, compared to the in vitro cell culture assay. It is concluded that represents a contribution to establish alternatives to monitor stability, quality control and thereby assure therapeutic efficacy of the biotechnology-derived medicine.Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorA insulina humana é um hormônio secretado pelas células β, das ilhotas de Langerhans, e regula os níveis sanguíneos de glicose. A insulina glargina é produzida pela tecnologia do DNA recombinante, expressa em Escherichia coli, e difere da insulina humana pela modificação da aspargina na posição 21 da cadeia A por uma glicina, além da adição de dois resíduos de arginina C-terminais na cadeia B. No presente trabalho foram desenvolvidos e validados métodos por cromatografia líquida em fase reversa (CL-FR) e por exclusão molecular (CL-EM) para a avaliação de insulina glargina em formulações biofarmacêuticas. No método por CL-FR, foi utilizada coluna Júpiter C4 (250 mm x 4,6 mm d.i.), mantida a 30 ºC. A fase móvel A foi constituída por tampão sulfato de sódio 0,05 M, pH 2,5, e a fase móvel B por acetonitrila, eluídas com fluxo constante de 0,5 mL/min. No método por CL-EM foi utilizada coluna BioSep-SEC-S 2000 (300 mm x 7.8 mm d.i.), mantida a 25 ºC. A fase móvel foi constituída de tampão MES 0,03 M, pH 2,5, eluída em vazão isocrática de 0,6 mL/min. Para ambos os métodos utilizou-se detector de arranjo de diodos (DAD) com detecções em 214 nm e 200 nm para CL-FR e CL-EM, respectivamente. A separação cromatográfica foi obtida nos tempos de 7,5 e 9,9 min, sendo linear na faixa de concentração de 0,05 - 200 μg/mL (R2 = 0,9998) e 0,02 - 180 μg/mL (R2 = 0,9999), respectivamente, para os métodos por CL-FR e CL-EM. Os limites de detecção e quantificação foram 0,018 e 0,054 μg/mL, respectivamente, para o método por CL-FR e 0,009 e 0,027 μg/mL por CL-EM. A especificidade foi avaliada em estudos de degradação, que também demonstraram que não houve interferência dos excipientes. A exatidão foi 100,13 e 99,38%, com bias inferior a 0,85 e 0,86%, respectivamente, para os métodos por CL-FR e CL-EM. Os métodos propostos foram aplicados para avaliação da potência de insulina glargina, de proteínas relacionadas agregados de alta massa molecular em formulações biofarmacêuticas, e os resultados foram comparados com o bioensaio por cultura de células in vitro, observando-se diferenças das médias de teor/potência 0,21% e 0,16% inferiores para os métodos por CL-FR e CL-EM. Concluí-se que representa contribuição para estabelecer procedimentos para monitorar a estabilidade, o controle da qualidade, garantindo a segurança e eficácia terapêutica do produto biotecnológico.Universidade Federal de Santa MariaBRAnálises Clínicas e ToxicológicasUFSMPrograma de Pós-Graduação em Ciências FarmacêuticasDalmora, Sergio Luizhttp://lattes.cnpq.br/4505166045049607Macedo, Rui Oliveirahttp://lattes.cnpq.br/8326594695097434Codevilla, Cristiane Francohttp://lattes.cnpq.br/3165544867590900Schramm, Vanessa Grigoletto2016-08-302016-08-302016-03-30info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfapplication/pdfSCHRAMM, Vanessa Grigoletto. Development and validation of chromatographic methods for the content/potency evaluation of insulin glargine. 2016. 61 f. Dissertação (Mestrado em Farmacologia) - Universidade Federal de Santa Maria, Santa Maria, 2016.http://repositorio.ufsm.br/handle/1/6037porinfo:eu-repo/semantics/openAccessreponame:Manancial - Repositório Digital da UFSMinstname:Universidade Federal de Santa Maria (UFSM)instacron:UFSM2022-05-02T13:56:06Zoai:repositorio.ufsm.br:1/6037Biblioteca Digital de Teses e Dissertaçõeshttps://repositorio.ufsm.br/ONGhttps://repositorio.ufsm.br/oai/requestatendimento.sib@ufsm.br||tedebc@gmail.comopendoar:2022-05-02T13:56:06Manancial - Repositório Digital da UFSM - Universidade Federal de Santa Maria (UFSM)false |
dc.title.none.fl_str_mv |
Desenvolvimento e validação de métodos cromatográficos para avaliação de teor/potência de insulina glargina Development and validation of chromatographic methods for the content/potency evaluation of insulin glargine |
title |
Desenvolvimento e validação de métodos cromatográficos para avaliação de teor/potência de insulina glargina |
spellingShingle |
Desenvolvimento e validação de métodos cromatográficos para avaliação de teor/potência de insulina glargina Schramm, Vanessa Grigoletto Insulina glargina Cromatografia líquida Validação Bioensaio Correlação Insulin glargine Liquid chromatography methods Validation Bioassay Correlation CNPQ::CIENCIAS DA SAUDE::FARMACIA |
title_short |
Desenvolvimento e validação de métodos cromatográficos para avaliação de teor/potência de insulina glargina |
title_full |
Desenvolvimento e validação de métodos cromatográficos para avaliação de teor/potência de insulina glargina |
title_fullStr |
Desenvolvimento e validação de métodos cromatográficos para avaliação de teor/potência de insulina glargina |
title_full_unstemmed |
Desenvolvimento e validação de métodos cromatográficos para avaliação de teor/potência de insulina glargina |
title_sort |
Desenvolvimento e validação de métodos cromatográficos para avaliação de teor/potência de insulina glargina |
author |
Schramm, Vanessa Grigoletto |
author_facet |
Schramm, Vanessa Grigoletto |
author_role |
author |
dc.contributor.none.fl_str_mv |
Dalmora, Sergio Luiz http://lattes.cnpq.br/4505166045049607 Macedo, Rui Oliveira http://lattes.cnpq.br/8326594695097434 Codevilla, Cristiane Franco http://lattes.cnpq.br/3165544867590900 |
dc.contributor.author.fl_str_mv |
Schramm, Vanessa Grigoletto |
dc.subject.por.fl_str_mv |
Insulina glargina Cromatografia líquida Validação Bioensaio Correlação Insulin glargine Liquid chromatography methods Validation Bioassay Correlation CNPQ::CIENCIAS DA SAUDE::FARMACIA |
topic |
Insulina glargina Cromatografia líquida Validação Bioensaio Correlação Insulin glargine Liquid chromatography methods Validation Bioassay Correlation CNPQ::CIENCIAS DA SAUDE::FARMACIA |
description |
hans, and is secreted into the bloodstream. It plays and important role in regulating the metabolic activities of the body, particularly the homeostasis of the blood glucose. Insulin glargine is a recombinant human insulin analogue produced by DNA technology using a strain of Escherichia coli and the insulin glargine differ only by three amino acids from human insulin. Reversed-phase liquid chromatography (RP-LC) and size exclusion liquid chromatography (SE-LC) methods were developed and validated for the assessment of insulin glargine in biopharmaceutical formulations. A RP-LC method was carried out on a Jupiter C4 column (250 mm x 4.6 mm i.d.), maintained at 30 ºC. The mobile phase A consisted of 0.05 M sodium sulphate buffer, pH 2.5, and the mobile phase B was acetonitrile. The SE-LC method was carried out on a BioSep-SEC-S 2000 column (300 mm x 7.8 mm i.d.), maintained at 25 ºC. The mobile phase consisted of 0.03 M MES acid buffer, pH 2.5, run isocratically at a flow rate of 0.6 mL/min. Chromatographic separation was obtained with retention times of 7.5 min, and 9.9 min, and was linear over the concentration range of 0.05 - 200 μg/mL (R2 = 0.9998) and 0.02 - 180 μg/mL (R2 = 0.9999), respectively, for RP-LC and SE-LC, with photodiode array (PDA) detection at 214 nm and 200 nm for RP-LC and SE-LC methods, respectively. The limits of detection and quantitation were 0.018 and 0.054 μg/mL, respectively, for the RP-LC and 0.009 and 0.027 μg/mL, for the SE-LC. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. Equally, the accuracy was 100.13% and 99.38%, with bias lower than 0.85% and than 0.86%. The validated methods were applied for the determination of insulin glargine and related proteins and high molecular mass, in biotechnology-derived products, giving lower mean differences of the estimated content/potencies of 0.21% and 0.16% for the RP-LC and SE-LC related, compared to the in vitro cell culture assay. It is concluded that represents a contribution to establish alternatives to monitor stability, quality control and thereby assure therapeutic efficacy of the biotechnology-derived medicine. |
publishDate |
2016 |
dc.date.none.fl_str_mv |
2016-08-30 2016-08-30 2016-03-30 |
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 |
SCHRAMM, Vanessa Grigoletto. Development and validation of chromatographic methods for the content/potency evaluation of insulin glargine. 2016. 61 f. Dissertação (Mestrado em Farmacologia) - Universidade Federal de Santa Maria, Santa Maria, 2016. http://repositorio.ufsm.br/handle/1/6037 |
identifier_str_mv |
SCHRAMM, Vanessa Grigoletto. Development and validation of chromatographic methods for the content/potency evaluation of insulin glargine. 2016. 61 f. Dissertação (Mestrado em Farmacologia) - Universidade Federal de Santa Maria, Santa Maria, 2016. |
url |
http://repositorio.ufsm.br/handle/1/6037 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal de Santa Maria BR Análises Clínicas e Toxicológicas UFSM Programa de Pós-Graduação em Ciências Farmacêuticas |
publisher.none.fl_str_mv |
Universidade Federal de Santa Maria BR Análises Clínicas e Toxicológicas UFSM Programa de Pós-Graduação em Ciências Farmacêuticas |
dc.source.none.fl_str_mv |
reponame:Manancial - Repositório Digital da UFSM instname:Universidade Federal de Santa Maria (UFSM) instacron:UFSM |
instname_str |
Universidade Federal de Santa Maria (UFSM) |
instacron_str |
UFSM |
institution |
UFSM |
reponame_str |
Manancial - Repositório Digital da UFSM |
collection |
Manancial - Repositório Digital da UFSM |
repository.name.fl_str_mv |
Manancial - Repositório Digital da UFSM - Universidade Federal de Santa Maria (UFSM) |
repository.mail.fl_str_mv |
atendimento.sib@ufsm.br||tedebc@gmail.com |
_version_ |
1805922050619998208 |