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
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/33653
Ver los metadatos del registro completo
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oai:ri.conicet.gov.ar:11336/33653 |
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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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1844613527681105920 |
score |
13.070432 |