Manufacturing of CAR-T cells using in-house vectors and the CliniMACS Prodigy
DOI:
https://doi.org/10.46765/2675-374X.2025v7n1e338Keywords:
CAR-T, In-house vectors, CliniMACS Prodigy, Quality controlAbstract
Objectives: This article provides a practical, regulatory-focused guide for the clinical-grade manufacturing of autologous CAR-T cells using in-house lentiviral vectors and the CliniMACS Prodigy system. Methods: A narrative review and technical synthesis of the scientific literature, institutional manufacturing workflows, platform-specific protocols, and Brazilian and international regulatory documents were performed. Results: The detailed process includes leukapheresis, receipt of the material, chain-of-identity and chain-of-custody controls, and initial testing of the starting material, including total nucleated cell count, CD3+ cell content, viability, and microbiological screening. The Prodigy system utilizes automated modules for magnetic T-cell enrichment, CD3/CD28 activation, lentiviral transduction, cell expansion, harvesting, formulation, and cryopreservation. Operational conduct can affect reproducibility, starting material quality, transduction efficiency, and the cell culture process. Final product release is conditional on comprehensive quality control, and coordination among the laboratories is essential. Accordingly, quality assurance procedures, deviation and corrective and preventive action management, process validation, and traceability constitute essential components in a manufacturing center. Conclusion: Integrating manufacturing and quality procedures within a structured workflow facilitates platform standardization and automation, hence promoting consistent CAR-T-cell production.
References
1. Mukherjee S, Reddy O, Panch S, Stroncek D. Establishment of a cell processing laboratory to support hematopoietic stem cell transplantation and chimeric antigen receptor (CAR)-T cell therapy. Transfus Apher Sci. 2021;60(1):103066. https://doi.org/10.1016/j.transci.2021.103066 DOI: https://doi.org/10.1016/j.transci.2021.103066
2. June CH, Sadelain M. Chimeric antigen receptor therapy. N Engl J Med. 2018;379(1):64-73. https://doi.org/10.1056/NEJMra1706169 DOI: https://doi.org/10.1056/NEJMra1706169
3. Zugasti I, Espinosa-Aroca L, Fidyt K, Mulens-Arias V, Diaz-Beya M, Juan M, et al. CAR-T cell therapy for cancer: current challenges and future directions. Signal Transduct Target Ther. 2025;10(1):210. https://doi.org/10.1038/s41392-025-02269-w DOI: https://doi.org/10.1038/s41392-025-02269-w
4. Levine BL, Miskin J, Wonnacott K, Keir C. Global manufacturing of CAR-T cell therapy. Mol Ther Methods Clin Dev. 2017;4:92-101. https://doi.org/10.1016/j.omtm.2016.12.006 DOI: https://doi.org/10.1016/j.omtm.2016.12.006
5. Abou-El-Enein M, Elsallab M, Feldman SA, Fesnak AD, Heslop HE, Marks P, et al. Scalable manufacturing of CAR-T cells for cancer immunotherapy. Blood Cancer Discov. 2021;2(5):408-422. https://doi.org/10.1158/2643-3230.BCD-21-0084 DOI: https://doi.org/10.1158/2643-3230.BCD-21-0084
6. Poorebrahim M, Sadeghi S, Fakhr E, Abazari MF, Poortahmasebi V, Kheirollahi A, et al. Production of CAR-T-cells by GMP-grade lentiviral vectors: latest advances and future prospects. Crit Rev Clin Lab Sci. 2019;56(6):393-419. https://doi.org/10.1080/10408363.2019.1633512 DOI: https://doi.org/10.1080/10408363.2019.1633512
7. Milone MC, O’Doherty U. Clinical use of lentiviral vectors. Leukemia. 2018;32(7):1529-1541. https://doi.org/10.1038/s41375-018-0106-0 DOI: https://doi.org/10.1038/s41375-018-0106-0
8. Brasil. Agência Nacional de Vigilância Sanitária. Resolução da Diretoria Colegiada - RDC nº 505, de 27 de maio de 2021. [cited 2026 May 25]. Available from: https://www.in.gov.br/en/web/dou/-/resolucao-rdc-n-505-de-27-de-maio-de-2021-323002775
9. Brasil. Agência Nacional de Vigilância Sanitária. Resolução da Diretoria Colegiada - RDC nº 506, de 27 de maio de 2021. [cited 2026 May 25]. Available from: https://www.in.gov.br/en/web/dou/-/resolucao-rdc-n-506-de-27-de-maio-de-2021-323008725
10. Brasil. Agência Nacional de Vigilância Sanitária. Resolução da Diretoria Colegiada - RDC nº 508, de 27 de maio de 2021. [cited 2026 May 25].
11. Mesquita FP, Maia MS, Souza PFN, Carvalho LEM, Carlos LMB, Duarte FB. Regulatory landscape for CAR-T therapy development in the Brazilian context. Expert Rev Anticancer Ther. 2026;26(1):1-18. https://doi.org/10.1080/14737140.2026.2617956 DOI: https://doi.org/10.1080/14737140.2026.2617956
12. Noaks E, Peticone C, Kotsopoulou E, Bracewell DG. Enriching leukapheresis improves T cell activation and transduction efficiency during CAR-T processing. Mol Ther Methods Clin Dev. 2021;20:675-687. https://doi.org/10.1016/j.omtm.2021.02.002 DOI: https://doi.org/10.1016/j.omtm.2021.02.002
13. Baguet C, Larghero J, Mebarki M. Early predictive factors of failure in autologous CAR-T-cell manufacturing and/or efficacy in hematologic malignancies. Blood Adv. 2024;8(2):337-342. https://doi.org/10.1182/bloodadvances.2023011992 DOI: https://doi.org/10.1182/bloodadvances.2023011992
14. Reddy OL, Stroncek DF, Panch SR. Improving CAR-T cell therapy by optimizing critical quality attributes. Semin Hematol. 2020;57(2):33-38. https://doi.org/10.1053/j.seminhematol.2020.07.005 DOI: https://doi.org/10.1053/j.seminhematol.2020.07.005
15. Brasil. Agência Nacional de Vigilância Sanitária. Resolução da Diretoria Colegiada - RDC nº 836, de 13 de dezembro de 2023. [cited 2026 May 25]. Available from: https://www.gov.br/anvisa/pt-br/assuntos/sangue/inspecao/arquivos/roteiro-comentado-rdc836_2023_v1.pdf
16. Marton C, Clémenceau B, Dachy G, Demerle C, Derenne S, Ferrand C, et al. Harmonisation of quality control tests for academic production of CAR-T cells: a position paper from the WP-bioproduction of the UNITC consortium. Bone Marrow Transplant. 2025;60(9):1209-1217. https://doi.org/10.1038/s41409-025-02637-8 DOI: https://doi.org/10.1038/s41409-025-02637-8
17. Wang X, Rivière I. Clinical manufacturing of CAR-T cells: foundation of a promising therapy. Mol Ther Oncolytics. 2016;3:16015. https://doi.org/10.1038/mto.2016.15 DOI: https://doi.org/10.1038/mto.2016.15
18. von der Leyen H, Delgado J, Mazouz C, Schmitt M, Caplan V. Implementation of a quality management system for decentralized manufacturing of cell and gene therapy products: technical and regulatory considerations. Front Med (Lausanne). 2025;12:1591751. https://doi.org/10.3389/fmed.2025.1591751 DOI: https://doi.org/10.3389/fmed.2025.1591751
19. Mock U, Nickolay L, Philip B, Cheung GW, Zhan H, Johnston ICD, et al. Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS Prodigy. Cytotherapy. 2016;18(8):1002-1011. https://doi.org/10.1016/j.jcyt.2016.05.009 DOI: https://doi.org/10.1016/j.jcyt.2016.05.009
20. Jackson Z, Roe A, Sharma AA, Lopes FBTP, Talla A, Kleinsorge-Block S, et al. Automated manufacture of autologous CD19 CAR-T cells for treatment of non-Hodgkin lymphoma. Front Immunol. 2020;11:1941. https://doi.org/10.3389/fimmu.2020.01941 DOI: https://doi.org/10.3389/fimmu.2020.01941
21. Ayala Ceja M, Khericha M, Harris CM, Puig-Saus C, Chen YY. CAR-T cell manufacturing: major process parameters and next-generation strategies. J Exp Med. 2024;221(2):e20230903. https://doi.org/10.1084/jem.20230903 DOI: https://doi.org/10.1084/jem.20230903
22. van der Walle CF, Godbert S, Saito G, Azhari Z. Formulation considerations for autologous T cell drug products. Pharmaceutics. 2021;13(8):1317. https://doi.org/10.3390/pharmaceutics13081317 DOI: https://doi.org/10.3390/pharmaceutics13081317
23. Palani HK, Arunachalam AK, Yasar M, Venkatraman A, Kulkarni U, Lionel SA, et al. Decentralized manufacturing of anti-CD19 CAR-T cells using CliniMACS Prodigy®: real-world experience and cost analysis in India. Bone Marrow Transplant. 2023;58(2):160-167. https://doi.org/10.1038/s41409-022-01866-5 DOI: https://doi.org/10.1038/s41409-022-01866-5
24. Simon CG Jr, Bozenhardt EH, Celluzzi CM, Dobnik D, Grant ML, Lakshmipathy U, et al. Analysis of the measurements used as potency tests for the 31 US FDA-approved cell therapy products. J Transl Med. 2025;23(1):259. https://doi.org/10.1186/s12967-025-06253-4 DOI: https://doi.org/10.1186/s12967-025-06253-4
25. Shi J, Yang J, Zheng Y, Wong PHH, Hu H, Ung COL. The clinical quality management system of advanced therapy medicinal products in the hospital setting: a scoping review. Mol Ther Methods Clin Dev. 2025;33:101485. https://doi.org/10.1016/j.omtm.2025.101485 DOI: https://doi.org/10.1016/j.omtm.2025.101485
26. U.S. Food and Drug Administration. Chemistry, manufacturing, and control (CMC) information for human gene therapy investigational new drug applications (INDs): guidance for industry. [cited 2026 May 25]. Available from: https://www.fda.gov/media/113760/download
27. Increasing production efficiency through electronic batch record systems: a case study. In: Camarinha-Matos LM, editor. Smart Innovation, Systems and Technologies. Vol. 52. Cham: Springer International Publishing; 2016. p. 261-269. https://doi.org/10.1007/978-3-319-32098-4_23 DOI: https://doi.org/10.1007/978-3-319-32098-4_23
28. Al Azawei A, Loughrey K, Surim K, Connolly ME, Naughton BD. The management of good manufacturing practice (GMP) inspections: a scoping review of the evidence. Front Med (Lausanne). 2025;12:1687864. https://doi.org/10.3389/fmed.2025.1687864 DOI: https://doi.org/10.3389/fmed.2025.1687864
29. Foundation for the Accreditation of Cellular Therapy; Joint Accreditation Committee ISCT-Europe & EBMT. FACT-JACIE international standards for hematopoietic cellular therapy product collection, processing, and administration. 9th ed. Version 9.1. Omaha (NE): FACT; 2025. [cited 2026 May 25]. Available from: https://www.ebmt.org/sites/default/files/2025-12/FACT-JACIE%20HCT%20Standards%209th%20Ed_V9-1.pdf
30. Arunagiri T, Kannaiah KP, Vasanthan M. Enhancing pharmaceutical product quality with a comprehensive corrective and preventive actions (CAPA) framework: from reactive to proactive. Cureus. 2024;16(9):e69762. https://doi.org/10.7759/cureus.69762 DOI: https://doi.org/10.7759/cureus.69762
31. Viganò M, Budelli S, Lavazza C, Montemurro T, Montelatici E, de Cesare S, et al. Tips and tricks for validation of quality control analytical methods in good manufacturing practice mesenchymal stromal cell production. Stem Cells Int. 2018;2018:3038565. https://doi.org/10.1155/2018/3038565 DOI: https://doi.org/10.1155/2018/3038565
32. Lavazza C, Budelli S, Montelatici E, Viganò M, Ulbar F, Catani L, et al. Process development and validation of expanded regulatory T cells for prospective applications: an example of manufacturing a personalized advanced therapy medicinal product. J Transl Med. 2022;20(1):14. https://doi.org/10.1186/s12967-021-03200-x DOI: https://doi.org/10.1186/s12967-021-03200-x
33. Delgado J, Roddie C, Schmitt M. Point-of-care production of CAR-T cells. In: Kröger N, Gribben J, Chabannon C, Yakoub-Agha I, Einsele H, editors. The EBMT/EHA CAR-T Cell Handbook. Cham: Springer; 2022. p. 45-49. https://doi.org/10.1007/978-3-030-94353-0_8 DOI: https://doi.org/10.1007/978-3-030-94353-0_8
34. Mitra A, Barua A, Huang L, Ganguly S, Feng Q, He B. From bench to bedside: the history and progress of CAR-T cell therapy. Front Immunol. 2023;14:1188049. https://doi.org/10.3389/fimmu.2023.1188049 DOI: https://doi.org/10.3389/fimmu.2023.1188049
35. Balke-Want H, Keerthi V, Cadinanos-Garai A, Fowler C, Gkitsas N, Brown AK, et al. Non-viral chimeric antigen receptor (CAR) T cells going viral. Immunooncol Technol. 2023;18:100375. https://doi.org/10.1016/j.iotech.2023.100375 DOI: https://doi.org/10.1016/j.iotech.2023.100375
36. Lei T, Wang Y, Zhang Y, Yang Y, Cao J, Huang J, et al. Leveraging CRISPR gene editing technology to optimize the efficacy, safety and accessibility of CAR-T-cell therapy. Leukemia. 2024;38(12):2517-2543. https://doi.org/10.1038/s41375-024-02444-y DOI: https://doi.org/10.1038/s41375-024-02444-y
37. Gupta D, Shaz B. Surveying local CAR-T-cell manufacturing processes to facilitate standardization and expand accessibility. J Transl Med. 2025;23(1):507. https://doi.org/10.1186/s12967-025-06400-x DOI: https://doi.org/10.1186/s12967-025-06400-x
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Copyright (c) 2026 Lucila Nassif Kerbauy, Felipe Pantoja Mesquita
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Conselho Nacional de Desenvolvimento Científico e Tecnológico
Grant numbers 311190/2026-0
