High yield process for the production of active human alpha-Galactosidase A in CHO-K1 cells through lentivirus transgenesis

Autores
Rodríguez, María Celeste; Ceaglio, Natalia Analia; Antuña, Sebastián; Tardivo, María Belén; Etcheverrigaray, Marina; Prieto, Claudio
Año de publicación
2017
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Fabry disease is an X-linked recessive disorder caused by a deficiency in lysosomal α-Galactosidase A. Currently, two enzyme replacement therapies (ERT) are available. However, access to orphan drugs continues to be limited by their high price. Selection of adequate high-expression systems still constitutes a challenge for alleviating the cost of treatments. Several strategies have been implemented, with varying success, trying to optimize the production process of recombinant human α-Galactosidase A (rhαGAL) in Chinese hamster ovary (CHO-K1) cells. Herein, we describe for the first time the application of a strategy based on third-generation lentiviral particles (LP) transduction of suspension CHO-K1 cells to obtain high-producing rhαGAL clones (3.5 to 59.4 pg.cell-1.d-1). After two purification steps, the active enzyme was recovered (2.4 x 106 U.mg-1) with 98% purity and 60% overall yield. Michaelis-Menten analysis demonstrated that rhαGAL was capable of hydrolyzing the synthetic substrate 4MU-α-Gal at a comparable rate to Fabrazyme®, the current CHO-derived ERT available for Fabry disease. In addition, rhαGAL presented the same mannose-6-phosphate (M6P) content, about 40% higher acid sialic amount and 33% reduced content of the immunogenic type of sialic acid (Neu5Gc) than the corresponding ones for Fabrazyme®. In comparison with other rhαGAL production processes reported to date, our approach achieves the highest rhαGAL productivity preserving adequate activity and glycosylation pattern. Even more, considering the improved glycosylation characteristics of rhαGAL, which might provide advantages regarding pharmacokinetics, our enzyme could be postulated as a promising alternative for therapeutic use in Fabry disease.
Fil: Rodríguez, María Celeste. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina
Fil: Ceaglio, Natalia Analia. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina
Fil: Antuña, Sebastián. Zeltek S.A.; Argentina
Fil: Tardivo, María Belén. Zeltek S.A.; Argentina
Fil: Etcheverrigaray, Marina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina
Fil: Prieto, Claudio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina
Materia
Rhalphagal
Fabry Disease
Lentiviral Particles (Lp)
Suspension Cho-K1
Glycosylation
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/33653

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repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling High yield process for the production of active human alpha-Galactosidase A in CHO-K1 cells through lentivirus transgenesisRodríguez, María CelesteCeaglio, Natalia AnaliaAntuña, SebastiánTardivo, María BelénEtcheverrigaray, MarinaPrieto, ClaudioRhalphagalFabry DiseaseLentiviral Particles (Lp)Suspension Cho-K1Glycosylationhttps://purl.org/becyt/ford/3.4https://purl.org/becyt/ford/3Fabry disease is an X-linked recessive disorder caused by a deficiency in lysosomal α-Galactosidase A. Currently, two enzyme replacement therapies (ERT) are available. However, access to orphan drugs continues to be limited by their high price. Selection of adequate high-expression systems still constitutes a challenge for alleviating the cost of treatments. Several strategies have been implemented, with varying success, trying to optimize the production process of recombinant human α-Galactosidase A (rhαGAL) in Chinese hamster ovary (CHO-K1) cells. Herein, we describe for the first time the application of a strategy based on third-generation lentiviral particles (LP) transduction of suspension CHO-K1 cells to obtain high-producing rhαGAL clones (3.5 to 59.4 pg.cell-1.d-1). After two purification steps, the active enzyme was recovered (2.4 x 106 U.mg-1) with 98% purity and 60% overall yield. Michaelis-Menten analysis demonstrated that rhαGAL was capable of hydrolyzing the synthetic substrate 4MU-α-Gal at a comparable rate to Fabrazyme®, the current CHO-derived ERT available for Fabry disease. In addition, rhαGAL presented the same mannose-6-phosphate (M6P) content, about 40% higher acid sialic amount and 33% reduced content of the immunogenic type of sialic acid (Neu5Gc) than the corresponding ones for Fabrazyme®. In comparison with other rhαGAL production processes reported to date, our approach achieves the highest rhαGAL productivity preserving adequate activity and glycosylation pattern. Even more, considering the improved glycosylation characteristics of rhαGAL, which might provide advantages regarding pharmacokinetics, our enzyme could be postulated as a promising alternative for therapeutic use in Fabry disease.Fil: Rodríguez, María Celeste. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Ceaglio, Natalia Analia. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Antuña, Sebastián. Zeltek S.A.; ArgentinaFil: Tardivo, María Belén. Zeltek S.A.; ArgentinaFil: Etcheverrigaray, Marina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Prieto, Claudio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; ArgentinaAmerican Chemical Society2017-08info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/33653Rodríguez, María Celeste; Tardivo, María Belén; Ceaglio, Natalia Analia; Antuña, Sebastián; Etcheverrigaray, Marina; Prieto, Claudio; et al.; High yield process for the production of active human alpha-Galactosidase A in CHO-K1 cells through lentivirus transgenesis; American Chemical Society; Biotechnology Progress; 33; 5; 8-2017; 1334-13458756-7938CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1002/btpr.2538info:eu-repo/semantics/altIdentifier/url/http://onlinelibrary.wiley.com/doi/10.1002/btpr.2538/abstract;jsessionid=8482CDD1AA5568B4B29792926906BA84.f01t02info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:49:20Zoai:ri.conicet.gov.ar:11336/33653instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 09:49:20.568CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv High yield process for the production of active human alpha-Galactosidase A in CHO-K1 cells through lentivirus transgenesis
title High yield process for the production of active human alpha-Galactosidase A in CHO-K1 cells through lentivirus transgenesis
spellingShingle High yield process for the production of active human alpha-Galactosidase A in CHO-K1 cells through lentivirus transgenesis
Rodríguez, María Celeste
Rhalphagal
Fabry Disease
Lentiviral Particles (Lp)
Suspension Cho-K1
Glycosylation
title_short High yield process for the production of active human alpha-Galactosidase A in CHO-K1 cells through lentivirus transgenesis
title_full High yield process for the production of active human alpha-Galactosidase A in CHO-K1 cells through lentivirus transgenesis
title_fullStr High yield process for the production of active human alpha-Galactosidase A in CHO-K1 cells through lentivirus transgenesis
title_full_unstemmed High yield process for the production of active human alpha-Galactosidase A in CHO-K1 cells through lentivirus transgenesis
title_sort High yield process for the production of active human alpha-Galactosidase A in CHO-K1 cells through lentivirus transgenesis
dc.creator.none.fl_str_mv Rodríguez, María Celeste
Ceaglio, Natalia Analia
Antuña, Sebastián
Tardivo, María Belén
Etcheverrigaray, Marina
Prieto, Claudio
author Rodríguez, María Celeste
author_facet Rodríguez, María Celeste
Ceaglio, Natalia Analia
Antuña, Sebastián
Tardivo, María Belén
Etcheverrigaray, Marina
Prieto, Claudio
author_role author
author2 Ceaglio, Natalia Analia
Antuña, Sebastián
Tardivo, María Belén
Etcheverrigaray, Marina
Prieto, Claudio
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv Rhalphagal
Fabry Disease
Lentiviral Particles (Lp)
Suspension Cho-K1
Glycosylation
topic Rhalphagal
Fabry Disease
Lentiviral Particles (Lp)
Suspension Cho-K1
Glycosylation
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.4
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv Fabry disease is an X-linked recessive disorder caused by a deficiency in lysosomal α-Galactosidase A. Currently, two enzyme replacement therapies (ERT) are available. However, access to orphan drugs continues to be limited by their high price. Selection of adequate high-expression systems still constitutes a challenge for alleviating the cost of treatments. Several strategies have been implemented, with varying success, trying to optimize the production process of recombinant human α-Galactosidase A (rhαGAL) in Chinese hamster ovary (CHO-K1) cells. Herein, we describe for the first time the application of a strategy based on third-generation lentiviral particles (LP) transduction of suspension CHO-K1 cells to obtain high-producing rhαGAL clones (3.5 to 59.4 pg.cell-1.d-1). After two purification steps, the active enzyme was recovered (2.4 x 106 U.mg-1) with 98% purity and 60% overall yield. Michaelis-Menten analysis demonstrated that rhαGAL was capable of hydrolyzing the synthetic substrate 4MU-α-Gal at a comparable rate to Fabrazyme®, the current CHO-derived ERT available for Fabry disease. In addition, rhαGAL presented the same mannose-6-phosphate (M6P) content, about 40% higher acid sialic amount and 33% reduced content of the immunogenic type of sialic acid (Neu5Gc) than the corresponding ones for Fabrazyme®. In comparison with other rhαGAL production processes reported to date, our approach achieves the highest rhαGAL productivity preserving adequate activity and glycosylation pattern. Even more, considering the improved glycosylation characteristics of rhαGAL, which might provide advantages regarding pharmacokinetics, our enzyme could be postulated as a promising alternative for therapeutic use in Fabry disease.
Fil: Rodríguez, María Celeste. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina
Fil: Ceaglio, Natalia Analia. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina
Fil: Antuña, Sebastián. Zeltek S.A.; Argentina
Fil: Tardivo, María Belén. Zeltek S.A.; Argentina
Fil: Etcheverrigaray, Marina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina
Fil: Prieto, Claudio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina
description Fabry disease is an X-linked recessive disorder caused by a deficiency in lysosomal α-Galactosidase A. Currently, two enzyme replacement therapies (ERT) are available. However, access to orphan drugs continues to be limited by their high price. Selection of adequate high-expression systems still constitutes a challenge for alleviating the cost of treatments. Several strategies have been implemented, with varying success, trying to optimize the production process of recombinant human α-Galactosidase A (rhαGAL) in Chinese hamster ovary (CHO-K1) cells. Herein, we describe for the first time the application of a strategy based on third-generation lentiviral particles (LP) transduction of suspension CHO-K1 cells to obtain high-producing rhαGAL clones (3.5 to 59.4 pg.cell-1.d-1). After two purification steps, the active enzyme was recovered (2.4 x 106 U.mg-1) with 98% purity and 60% overall yield. Michaelis-Menten analysis demonstrated that rhαGAL was capable of hydrolyzing the synthetic substrate 4MU-α-Gal at a comparable rate to Fabrazyme®, the current CHO-derived ERT available for Fabry disease. In addition, rhαGAL presented the same mannose-6-phosphate (M6P) content, about 40% higher acid sialic amount and 33% reduced content of the immunogenic type of sialic acid (Neu5Gc) than the corresponding ones for Fabrazyme®. In comparison with other rhαGAL production processes reported to date, our approach achieves the highest rhαGAL productivity preserving adequate activity and glycosylation pattern. Even more, considering the improved glycosylation characteristics of rhαGAL, which might provide advantages regarding pharmacokinetics, our enzyme could be postulated as a promising alternative for therapeutic use in Fabry disease.
publishDate 2017
dc.date.none.fl_str_mv 2017-08
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/33653
Rodríguez, María Celeste; Tardivo, María Belén; Ceaglio, Natalia Analia; Antuña, Sebastián; Etcheverrigaray, Marina; Prieto, Claudio; et al.; High yield process for the production of active human alpha-Galactosidase A in CHO-K1 cells through lentivirus transgenesis; American Chemical Society; Biotechnology Progress; 33; 5; 8-2017; 1334-1345
8756-7938
CONICET Digital
CONICET
url http://hdl.handle.net/11336/33653
identifier_str_mv Rodríguez, María Celeste; Tardivo, María Belén; Ceaglio, Natalia Analia; Antuña, Sebastián; Etcheverrigaray, Marina; Prieto, Claudio; et al.; High yield process for the production of active human alpha-Galactosidase A in CHO-K1 cells through lentivirus transgenesis; American Chemical Society; Biotechnology Progress; 33; 5; 8-2017; 1334-1345
8756-7938
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1002/btpr.2538
info:eu-repo/semantics/altIdentifier/url/http://onlinelibrary.wiley.com/doi/10.1002/btpr.2538/abstract;jsessionid=8482CDD1AA5568B4B29792926906BA84.f01t02
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv American Chemical Society
publisher.none.fl_str_mv American Chemical Society
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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