Development of recombinant human granulocyte colony-stimulating factor (nartograstim) production process in Escherichia coli compatible with industrial scale and with no antibiotics in the culture medium

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dc.contributorLEDV - Laboratório de Desenvolvimento de Vacinaspt_BR
dc.contributorLab. Bacteriologiapt_BR
dc.contributorCentro Bioindustrialpt_BR
dc.contributor.authorEguia, Fara Amelia Primellespt_BR
dc.contributor.authorMascarelli, Daniele Enriquettopt_BR
dc.contributor.authorCarvalho, Eneaspt_BR
dc.contributor.authorRodríguez, Gretel R.pt_BR
dc.contributor.authorMakiyama, Edsonpt_BR
dc.contributor.authorBorelli, Primaverapt_BR
dc.contributor.authorLiberman, Celiapt_BR
dc.contributor.authorHo, Paulo Leept_BR
dc.contributor.authorBarazzone, Giovana Cappiopt_BR
dc.contributor.authorGonçalves, Viviane Maimonipt_BR
dc.date.accessioned2020-12-04T18:19:18Z-
dc.date.available2020-12-04T18:19:18Z-
dc.date.issued2021pt_BR
dc.identifier.citationEguia FAP, Mascarelli DE, Carvalho E, Rodríguez GR., Makiyama E, Borelli P, et al. Development of recombinant human granulocyte colony-stimulating factor (nartograstim) production process in Escherichia coli compatible with industrial scale and with no antibiotics in the culture medium. Appl. Microbiol. Biotechnol.. 2021 Nov;105:169-183. doi:10.1007/s00253-020-11014-y.pt_BR
dc.identifier.urihttps://repositorio.butantan.gov.br/handle/butantan/3345-
dc.description.abstractThe granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that has important clinical applications for treating neutropenia. Nartograstim is a recombinant variant of human G-CSF. Nartograstim has been produced in Escherichia coli as inclusion bodies (IB) and presents higher stability and biological activity than the wild type of human G-CSF because of its mutations. We developed a production process of nartograstim in a 10-L bioreactor using auto-induction or chemically defined medium. After cell lysis, centrifugation, IB washing, and IB solubilization, the following three refolding methods were evaluated: diafiltration, dialysis, and direct dilution in two refolding buffers. Western blot and SDS-PAGE confirmed the identity of 18.8-kDa bands as nartograstim in both cultures. The auto-induction medium produced 1.17 g/L and chemically defined medium produced 0.95 g/L. The dilution method yielded the highest percentage of refolding (99%). After refolding, many contaminant proteins precipitated during pH adjustment to 5.2, increasing purity from 50 to 78%. After applying the supernatant to cation exchange chromatography (CEC), nartograstim recovery was low and the purity was 87%. However, when the refolding solution was applied to anion exchange chromatography followed by CEC, 91%-98% purity and 2.2% recovery were obtained. The purification process described in this work can be used to obtain nartograstim with high purity, structural integrity, and the expected biological activity.pt_BR
dc.description.sponsorshipFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)pt_BR
dc.description.sponsorshipFundação Butantanpt_BR
dc.description.sponsorshipCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)pt_BR
dc.description.sponsorshipConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)pt_BR
dc.format.extent169-183pt_BR
dc.language.isoEnglishpt_BR
dc.relation.ispartofApplied Microbiology and Biotechnologypt_BR
dc.titleDevelopment of recombinant human granulocyte colony-stimulating factor (nartograstim) production process in Escherichia coli compatible with industrial scale and with no antibiotics in the culture mediumpt_BR
dc.typeArticlept_BR
dc.identifier.doi10.1007/s00253-020-11014-ypt_BR
dc.identifier.urlhttps://doi.org/10.1007/s00253-020-11014-ypt_BR
dc.contributor.externalUniversidade de São Paulo (USP)pt_BR
dc.identifier.citationvolume105pt_BR
dc.subject.keywordG-CSFpt_BR
dc.subject.keywordBioreactor cultivationpt_BR
dc.subject.keywordInclusion bodiespt_BR
dc.subject.keywordRefoldingpt_BR
dc.subject.keywordPurificationpt_BR
dc.subject.keywordRecoverypt_BR
dc.relation.ispartofabbreviatedAppl. Microbiol. Biotechnol.pt_BR
dc.identifier.citationabntv. 105, p. 169-183, nov. 2021pt_BR
dc.identifier.citationvancouver2021 Nov;105:169-183pt_BR
dc.contributor.butantanEguia, Fara Amelia Primelles|:Aluno|:LEDV - Laboratório de Desenvolvimento de Vacinas|:PrimeiroAutorpt_BR
dc.contributor.butantanMascarelli, Daniele Enriquetto|:Aluno|:LEDV - Laboratório de Desenvolvimento de Vacinaspt_BR
dc.contributor.butantanCarvalho, Eneas|:Pesquisador|:Lab. Bacteriologiapt_BR
dc.contributor.butantanLiberman, Celia|:Colaborador|:LEDV - Laboratório de Desenvolvimento de Vacinaspt_BR
dc.contributor.butantanHo, Paulo Lee|:Pesquisador|:Centro Bioindustrialpt_BR
dc.contributor.butantanBarazzone, Giovana Cappio|:Pesquisador|:LEDV - Laboratório de Desenvolvimento de Vacinaspt_BR
dc.contributor.butantanGonçalves, Viviane Maimoni|:Pesquisador|:LEDV - Laboratório de Desenvolvimento de Vacinas|:Autor de correspondênciapt_BR
dc.sponsorship.butantanFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)¦¦2018/10384-4pt_BR
dc.sponsorship.butantanFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)¦¦2016/50413-8pt_BR
dc.sponsorship.butantanCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)¦¦12585-13-0pt_BR
dc.sponsorship.butantanConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)¦¦171057/2018-0pt_BR
dc.identifier.bvsccBR78.1pt_BR
dc.identifier.bvsdbIBProdpt_BR
dc.description.dbindexedYespt_BR
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