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A scalable insect cell-based production process of the human recombinant BMX for in-vitro covalent ligand high-throughput screening

2020Journal article Restricted access
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dc.contributor.authorSousa, Bárbara B.
dc.contributor.authorSousa, Marcos F. Q.
dc.contributor.authorMarques, Marta C.
dc.contributor.authorSeixas, João D.
dc.contributor.authorBrito, José A.
dc.contributor.authorMatias, Pedro M.
dc.contributor.authorBernardes, Gonçalo J. L.
dc.contributor.authorRoldão, António
dc.date.accessioned2021-12-02T14:27:19Z
dc.date.available2021-12-02T14:27:19Z
dc.date.issued2020
dc.description© Springer-Verlag GmbH Germany, part of Springer Nature 2020pt_PT
dc.description.abstractBone Marrow Tyrosine kinase in the chromosome X (BMX) is a TEC family kinase associated with numerous pathological pathways in cancer cells. Covalent inhibition of BMX activity holds promise as a therapeutic approach against cancer. To screen for potent and selective covalent BMX inhibitors, large quantities of highly pure BMX are normally required which is challenging with the currently available production and purification processes. Here, we developed a scalable production process for the human recombinant BMX (hrBMX) using the insect cell-baculovirus expression vector system. Comparable expression levels were obtained in small-scale shake flasks (13 mL) and in stirred-tank bioreactors (STB, 5 L). A two-step chromatographic-based process was implemented, reducing purification times by 75% when compared to traditional processes, while maintaining hrBMX stability. The final production yield was 24 mg of purified hrBMX per litter of cell culture, with a purity of > 99%. Product quality was assessed and confirmed through a series of biochemical and biophysical assays, including circular dichroism and dynamic light scattering. Overall, the platform herein developed was capable of generating 100 mg purified hrBMX from 5 L STB in just 34 days, thus having the potential to assist in-vitro covalent ligand high-throughput screening for BMX activity inhibition.pt_PT
dc.description.sponsorshipThis work has received funding from the European Union Horizon 2020 research and innovation program, under grant agreement No 702428. Royal Society to G.J.L.B. (URF\R\180019), FCT Portugal (iFCT to G.J.L.B., IF/00624/2015, Postdoctoral Fellowship SFRH/BPD/95253/2013 to J.D.S., and PTDC/MED-QUI/28764/2017 to J.D.S.), Marie Sklodowska-Curie IF (grant agreement No. 702428 to J.D.S. Investigador FCT programme 2014 (IF/01704/2014 and IF/01704/2014/CP1229/CT0001) to A.R. and from iNOVA4 Health Research Unit (LISBOA-01-0145-FEDER-007344) FCT/ Ministério da Educação e Ciência, FCT/MCTES, FEDER, and Programa Operacional Competitividade e Internacionalização (POCI) are also acknowledged. FCT SFRH/BPD/118731/2016 to M.C.M. FCT SFRH/BD/143583/2019 to B.B.S.pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.citationBioprocess Biosyst Eng. 2021 Jan;44(1):209-215pt_PT
dc.identifier.doi10.1007/s00449-020-02421-6pt_PT
dc.identifier.eissn1615-7605
dc.identifier.issn1615-7591
dc.identifier.urihttp://hdl.handle.net/10451/50243
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherSpringer Naturept_PT
dc.relationIF/00624/2015pt_PT
dc.relationA Small Molecule Drug Conjugate SMDC for Prostate Cancer Therapy
dc.relationIF/01704/2014pt_PT
dc.relationNext Generation Seasonal Influenza Vaccines: Towards a VLP-based Production Platform
dc.relationEngineering fusion inhibitor peptides against childhood respiratory viruses
dc.relationCombinatorial Antibody Synthesis for the discovery of Targeted Immunomodulators
dc.relation.publisherversionhttps://www.springer.com/journal/449pt_PT
dc.subjectBioprocess developmentpt_PT
dc.subjectCancer therapypt_PT
dc.subjectIC-BEVSpt_PT
dc.subjecthrBMX crystallizationpt_PT
dc.subjecthrBMX productionpt_PT
dc.titleA scalable insect cell-based production process of the human recombinant BMX for in-vitro covalent ligand high-throughput screeningpt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardTitleA Small Molecule Drug Conjugate SMDC for Prostate Cancer Therapy
oaire.awardTitleNext Generation Seasonal Influenza Vaccines: Towards a VLP-based Production Platform
oaire.awardTitleEngineering fusion inhibitor peptides against childhood respiratory viruses
oaire.awardTitleCombinatorial Antibody Synthesis for the discovery of Targeted Immunomodulators
oaire.awardURIinfo:eu-repo/grantAgreement/FCT//SFRH%2FBPD%2F95253%2F2013/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/3599-PPCDT/PTDC%2FMED-QUI%2F28764%2F2017/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/Investigador FCT/IF%2F01704%2F2014%2FCP1229%2FCT0001/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT//SFRH%2FBPD%2F118731%2F2016/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/OE/SFRH%2FBD%2F143583%2F2019/PT
oaire.citation.endPage215pt_PT
oaire.citation.issue1pt_PT
oaire.citation.startPage209pt_PT
oaire.citation.titleBioprocess and Biosystems Engineeringpt_PT
oaire.citation.volume44pt_PT
oaire.fundingStream3599-PPCDT
oaire.fundingStreamInvestigador FCT
oaire.fundingStreamOE
person.familyNameC. Marques
person.familyNameBernardes
person.givenNameMarta
person.givenNameGonçalo
person.identifier.ciencia-id3A1D-10AB-43BC
person.identifier.orcid0000-0002-5093-5918
person.identifier.orcid0000-0001-6594-8917
person.identifier.ridA-5725-2019
person.identifier.scopus-author-id35085375600
person.identifier.scopus-author-id14046757500
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
rcaap.rightsrestrictedAccesspt_PT
rcaap.typearticlept_PT
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