A quality-by-design approach to improve process understanding and optimise the production and quality of CAR-T cells in automated stirred-tank bioreactors
Ex vivo genetically-modified cellular immunotherapies, such as chimeric antigen receptor T cell (CAR-T) therapies, have generated significant clinical and commercial outcomes due to their unparalleled response rates against relapsed and refractory blood cancers. However, the development and scalable...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2024-04-01
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| Series: | Frontiers in Immunology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2024.1335932/full |
| _version_ | 1797217043355795456 |
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| author | Tiffany Hood Fern Slingsby Viktor Sandner Winfried Geis Timo Schmidberger Nicola Bevan Quentin Vicard Julia Hengst Pierre Springuel Noushin Dianat Qasim A. Rafiq |
| author_facet | Tiffany Hood Fern Slingsby Viktor Sandner Winfried Geis Timo Schmidberger Nicola Bevan Quentin Vicard Julia Hengst Pierre Springuel Noushin Dianat Qasim A. Rafiq |
| author_sort | Tiffany Hood |
| collection | DOAJ |
| description | Ex vivo genetically-modified cellular immunotherapies, such as chimeric antigen receptor T cell (CAR-T) therapies, have generated significant clinical and commercial outcomes due to their unparalleled response rates against relapsed and refractory blood cancers. However, the development and scalable manufacture of these novel therapies remains challenging and further process understanding and optimisation is required to improve product quality and yield. In this study, we employ a quality-by-design (QbD) approach to systematically investigate the impact of critical process parameters (CPPs) during the expansion step on the critical quality attributes (CQAs) of CAR-T cells. Utilising the design of experiments (DOE) methodology, we investigated the impact of multiple CPPs, such as number of activations, culture seeding density, seed train time, and IL-2 concentration, on CAR-T CQAs including, cell yield, viability, metabolism, immunophenotype, T cell differentiation, exhaustion and CAR expression. Initial studies undertaken in G-Rex® 24 multi-well plates demonstrated that the combination of a single activation step and a shorter, 3-day, seed train resulted in significant CAR-T yield and quality improvements, specifically a 3-fold increase in cell yield, a 30% reduction in exhaustion marker expression and more efficient metabolism when compared to a process involving 2 activation steps and a 7-day seed train. Similar findings were observed when the CPPs identified in the G-Rex® multi-well plates studies were translated to a larger-scale automated, controlled stirred-tank bioreactor (Ambr® 250 High Throughput) process. The single activation step and reduced seed train time resulted in a similar, significant improvement in CAR-T CQAs including cell yield, quality and metabolism in the Ambr® 250 High Throughput bioreactor, thereby validating the findings of the small-scale studies and resulting in significant process understanding and improvements. This study provides a methodology for the systematic investigation of CAR-T CPPs and the findings demonstrate the scope and impact of enhanced process understanding for improved CAR-T production. |
| first_indexed | 2024-04-24T11:55:35Z |
| format | Article |
| id | doaj.art-88a6db15b9b047b2b3a776a3de30ffb6 |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-04-24T11:55:35Z |
| publishDate | 2024-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-88a6db15b9b047b2b3a776a3de30ffb62024-04-09T04:52:20ZengFrontiers Media S.A.Frontiers in Immunology1664-32242024-04-011510.3389/fimmu.2024.13359321335932A quality-by-design approach to improve process understanding and optimise the production and quality of CAR-T cells in automated stirred-tank bioreactorsTiffany Hood0Fern Slingsby1Viktor Sandner2Winfried Geis3Timo Schmidberger4Nicola Bevan5Quentin Vicard6Julia Hengst7Pierre Springuel8Noushin Dianat9Qasim A. Rafiq10Department of Biochemical Engineering, University College London, London, United KingdomProduct Excellence Bioreactor Technology, Sartorius Stedim UK Limited, Epsom, United KingdomDigital Solutions, Sartorius Stedim Austria GmbH, Vienna, AustriaDigital Solutions, Sartorius Stedim Biotech GmbH, Goettingen, GermanyDigital Solutions, Sartorius Stedim Biotech GmbH, Goettingen, GermanyBioAnalytics Application Development, Essen BioScience Ltd. (Part of the Sartorius Group), Royston, United KingdomCell Culture Technology Marketing, Sartorius Stedim France S.A.S., Aubagne, FranceCell Culture Technology Marketing, Sartorius Stedim Biotech GmbH, Goettingen, GermanyDepartment of Biochemical Engineering, University College London, London, United KingdomCell Culture Technology Marketing, Sartorius Stedim France S.A.S., Aubagne, FranceDepartment of Biochemical Engineering, University College London, London, United KingdomEx vivo genetically-modified cellular immunotherapies, such as chimeric antigen receptor T cell (CAR-T) therapies, have generated significant clinical and commercial outcomes due to their unparalleled response rates against relapsed and refractory blood cancers. However, the development and scalable manufacture of these novel therapies remains challenging and further process understanding and optimisation is required to improve product quality and yield. In this study, we employ a quality-by-design (QbD) approach to systematically investigate the impact of critical process parameters (CPPs) during the expansion step on the critical quality attributes (CQAs) of CAR-T cells. Utilising the design of experiments (DOE) methodology, we investigated the impact of multiple CPPs, such as number of activations, culture seeding density, seed train time, and IL-2 concentration, on CAR-T CQAs including, cell yield, viability, metabolism, immunophenotype, T cell differentiation, exhaustion and CAR expression. Initial studies undertaken in G-Rex® 24 multi-well plates demonstrated that the combination of a single activation step and a shorter, 3-day, seed train resulted in significant CAR-T yield and quality improvements, specifically a 3-fold increase in cell yield, a 30% reduction in exhaustion marker expression and more efficient metabolism when compared to a process involving 2 activation steps and a 7-day seed train. Similar findings were observed when the CPPs identified in the G-Rex® multi-well plates studies were translated to a larger-scale automated, controlled stirred-tank bioreactor (Ambr® 250 High Throughput) process. The single activation step and reduced seed train time resulted in a similar, significant improvement in CAR-T CQAs including cell yield, quality and metabolism in the Ambr® 250 High Throughput bioreactor, thereby validating the findings of the small-scale studies and resulting in significant process understanding and improvements. This study provides a methodology for the systematic investigation of CAR-T CPPs and the findings demonstrate the scope and impact of enhanced process understanding for improved CAR-T production.https://www.frontiersin.org/articles/10.3389/fimmu.2024.1335932/fullimmunotherapyCAR-Tprocess understandingquality-by-designT cellsprocess optimisation |
| spellingShingle | Tiffany Hood Fern Slingsby Viktor Sandner Winfried Geis Timo Schmidberger Nicola Bevan Quentin Vicard Julia Hengst Pierre Springuel Noushin Dianat Qasim A. Rafiq A quality-by-design approach to improve process understanding and optimise the production and quality of CAR-T cells in automated stirred-tank bioreactors Frontiers in Immunology immunotherapy CAR-T process understanding quality-by-design T cells process optimisation |
| title | A quality-by-design approach to improve process understanding and optimise the production and quality of CAR-T cells in automated stirred-tank bioreactors |
| title_full | A quality-by-design approach to improve process understanding and optimise the production and quality of CAR-T cells in automated stirred-tank bioreactors |
| title_fullStr | A quality-by-design approach to improve process understanding and optimise the production and quality of CAR-T cells in automated stirred-tank bioreactors |
| title_full_unstemmed | A quality-by-design approach to improve process understanding and optimise the production and quality of CAR-T cells in automated stirred-tank bioreactors |
| title_short | A quality-by-design approach to improve process understanding and optimise the production and quality of CAR-T cells in automated stirred-tank bioreactors |
| title_sort | quality by design approach to improve process understanding and optimise the production and quality of car t cells in automated stirred tank bioreactors |
| topic | immunotherapy CAR-T process understanding quality-by-design T cells process optimisation |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2024.1335932/full |
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