Towards a scalable bioprocess for rAAV production using a HeLa stable cell line
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Publication Date: | 2023 |
Other Authors: | , , , , , , |
Format: | Article |
Language: | eng |
Source: | Repositórios Científicos de Acesso Aberto de Portugal (RCAAP) |
Download full: | http://hdl.handle.net/10362/165687 |
Summary: | Funding Information: We thank Dr. Maria João Sebastião, Dr. Mafalda Dias, Maria Gonçalves and Mariana Antunes for their helpful discussion and experimental support. This work was funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), the Associate Laboratory LS4FUTURE (LA/P/0087/2020) and project PTDC/BTM-ORG/1383/2020, and by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 813453 and TRANSVAC2 No. 730964; J.M.E. is funded by Stimulus of Scientific Employment, Individual Support program (2020.01216.CEECIND) from FCT; F.M is funded by PhD fellowship (2022.11494.BD) from FCT. Images were generated with Biorender.com. Funding Information: We thank Dr. Maria João Sebastião, Dr. Mafalda Dias, Maria Gonçalves and Mariana Antunes for their helpful discussion and experimental support. This work was funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), the Associate Laboratory LS4FUTURE (LA/P/0087/2020) and project PTDC/BTM‐ORG/1383/2020, and by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska‐Curie grant agreement No. 813453 and TRANSVAC2 No. 813453; J.M.E. is funded by Stimulus of Scientific Employment, Individual Support program (2020.01216.CEECIND) from FCT; F.M is funded by PhD fellowship (2022.11494.BD) from FCT. Images were generated with Biorender.com . Publisher Copyright: © 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC. |
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Towards a scalable bioprocess for rAAV production using a HeLa stable cell linebioprocess developmentcell line developmentdownstream processinggene therapyHeLaS3recombinant adeno-associated viruses (rAAV)stable cell lineBiotechnologyBioengineeringApplied Microbiology and BiotechnologyFunding Information: We thank Dr. Maria João Sebastião, Dr. Mafalda Dias, Maria Gonçalves and Mariana Antunes for their helpful discussion and experimental support. This work was funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), the Associate Laboratory LS4FUTURE (LA/P/0087/2020) and project PTDC/BTM-ORG/1383/2020, and by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 813453 and TRANSVAC2 No. 730964; J.M.E. is funded by Stimulus of Scientific Employment, Individual Support program (2020.01216.CEECIND) from FCT; F.M is funded by PhD fellowship (2022.11494.BD) from FCT. Images were generated with Biorender.com. Funding Information: We thank Dr. Maria João Sebastião, Dr. Mafalda Dias, Maria Gonçalves and Mariana Antunes for their helpful discussion and experimental support. This work was funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), the Associate Laboratory LS4FUTURE (LA/P/0087/2020) and project PTDC/BTM‐ORG/1383/2020, and by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska‐Curie grant agreement No. 813453 and TRANSVAC2 No. 813453; J.M.E. is funded by Stimulus of Scientific Employment, Individual Support program (2020.01216.CEECIND) from FCT; F.M is funded by PhD fellowship (2022.11494.BD) from FCT. Images were generated with Biorender.com . Publisher Copyright: © 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.The majority of recombinant adeno-associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale-up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa-based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems. Nevertheless, the time required to generate the producer cell lines combined with the complexity of rAAV production and purification processes still pose several barriers to the use of this platform as a suitable alternative to the HEK293 transient transfection. In this work we streamlined the cell line development and bioprocessing for the HeLaS3-based production of rAAV. By exploring this optimized approach, producer cell lines were generated in 3-4 months, and presented rAAV2 volumetric production (bulk) > 3 × 1011 vg/mL and full to empty capsids ratio (>70%) at 2 L bioreactor scale. Moreover, the established downstream process, based on ion exchange and affinity-based chromatography, efficiently eliminated process related impurities, including the Adenovirus 5 helper virus required for production with a log reduction value of 9. Overall, we developed a time-efficient and robust rAAV bioprocess using a stable producer cell line achieving purified rAAV2 yields > 1 × 1011 vg/mL. This optimized platform may address manufacturing challenges for rAAV based medicines.Instituto de Tecnologia Química e Biológica António Xavier (ITQB)RUNEscandell, JoséMoura, FilipaCarvalho, Sofia B.Silva, Ricardo J.S.Correia, RicardoRoldão, AntonioGomes-Alves, PatríciaAlves, Paula M.2024-04-01T23:17:45Z2023-092023-09-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article10application/pdfhttp://hdl.handle.net/10362/165687eng0006-3592PURE: 73157781https://doi.org/10.1002/bit.28394info:eu-repo/semantics/openAccessreponame:Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)instname:FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologiainstacron:RCAAP2024-05-22T18:20:05Zoai:run.unl.pt:10362/165687Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireinfo@rcaap.ptopendoar:https://opendoar.ac.uk/repository/71602025-05-28T17:50:50.825143Repositórios Científicos de Acesso Aberto de Portugal (RCAAP) - FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologiafalse |
dc.title.none.fl_str_mv |
Towards a scalable bioprocess for rAAV production using a HeLa stable cell line |
title |
Towards a scalable bioprocess for rAAV production using a HeLa stable cell line |
spellingShingle |
Towards a scalable bioprocess for rAAV production using a HeLa stable cell line Escandell, José bioprocess development cell line development downstream processing gene therapy HeLaS3 recombinant adeno-associated viruses (rAAV) stable cell line Biotechnology Bioengineering Applied Microbiology and Biotechnology |
title_short |
Towards a scalable bioprocess for rAAV production using a HeLa stable cell line |
title_full |
Towards a scalable bioprocess for rAAV production using a HeLa stable cell line |
title_fullStr |
Towards a scalable bioprocess for rAAV production using a HeLa stable cell line |
title_full_unstemmed |
Towards a scalable bioprocess for rAAV production using a HeLa stable cell line |
title_sort |
Towards a scalable bioprocess for rAAV production using a HeLa stable cell line |
author |
Escandell, José |
author_facet |
Escandell, José Moura, Filipa Carvalho, Sofia B. Silva, Ricardo J.S. Correia, Ricardo Roldão, Antonio Gomes-Alves, Patrícia Alves, Paula M. |
author_role |
author |
author2 |
Moura, Filipa Carvalho, Sofia B. Silva, Ricardo J.S. Correia, Ricardo Roldão, Antonio Gomes-Alves, Patrícia Alves, Paula M. |
author2_role |
author author author author author author author |
dc.contributor.none.fl_str_mv |
Instituto de Tecnologia Química e Biológica António Xavier (ITQB) RUN |
dc.contributor.author.fl_str_mv |
Escandell, José Moura, Filipa Carvalho, Sofia B. Silva, Ricardo J.S. Correia, Ricardo Roldão, Antonio Gomes-Alves, Patrícia Alves, Paula M. |
dc.subject.por.fl_str_mv |
bioprocess development cell line development downstream processing gene therapy HeLaS3 recombinant adeno-associated viruses (rAAV) stable cell line Biotechnology Bioengineering Applied Microbiology and Biotechnology |
topic |
bioprocess development cell line development downstream processing gene therapy HeLaS3 recombinant adeno-associated viruses (rAAV) stable cell line Biotechnology Bioengineering Applied Microbiology and Biotechnology |
description |
Funding Information: We thank Dr. Maria João Sebastião, Dr. Mafalda Dias, Maria Gonçalves and Mariana Antunes for their helpful discussion and experimental support. This work was funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), the Associate Laboratory LS4FUTURE (LA/P/0087/2020) and project PTDC/BTM-ORG/1383/2020, and by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 813453 and TRANSVAC2 No. 730964; J.M.E. is funded by Stimulus of Scientific Employment, Individual Support program (2020.01216.CEECIND) from FCT; F.M is funded by PhD fellowship (2022.11494.BD) from FCT. Images were generated with Biorender.com. Funding Information: We thank Dr. Maria João Sebastião, Dr. Mafalda Dias, Maria Gonçalves and Mariana Antunes for their helpful discussion and experimental support. This work was funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), the Associate Laboratory LS4FUTURE (LA/P/0087/2020) and project PTDC/BTM‐ORG/1383/2020, and by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska‐Curie grant agreement No. 813453 and TRANSVAC2 No. 813453; J.M.E. is funded by Stimulus of Scientific Employment, Individual Support program (2020.01216.CEECIND) from FCT; F.M is funded by PhD fellowship (2022.11494.BD) from FCT. Images were generated with Biorender.com . Publisher Copyright: © 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC. |
publishDate |
2023 |
dc.date.none.fl_str_mv |
2023-09 2023-09-01T00:00:00Z 2024-04-01T23:17:45Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/article |
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article |
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http://hdl.handle.net/10362/165687 |
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eng |
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