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Derenne, S., K. Tertrais, A. G. Chartois, F. Auffray, B. Clemenceau, and H. Vie. "Production de MTI de grade clinique par l’EFS Atlantic Bio GMP : transposition d’échelle, validation et production de lymphocytes T cytotoxiques anti-CMV tierce partie." Transfusion Clinique et Biologique 20, no. 3 (June 2013): 277. http://dx.doi.org/10.1016/j.tracli.2013.04.085.
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Rudiyanto, Heru. "The Study of Good Manufacturing Practices (GMP) and Good Quality Wingko Based on SNI-01-4311-1996." JURNAL KESEHATAN LINGKUNGAN 8, no. 2 (July 5, 2016): 148. http://dx.doi.org/10.20473/jkl.v8i2.2016.148-157.
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Abstract: Wingko is a special snack from Kecamatan Babat, Kabupaten Lamongan. One of home industries that produce Wingko in Babat City is UD. Bintang Jaya. The making process of Wingko is done traditionally, by using human power. It is really possible for microorganisms to contaminate and affect the quality of Wingko through this process. To raise the quality of food, food controlling is badly needed. Good Manufacturing Practices (GMP) is one of systems that describes the terms that have to be fulfi lled by home industries. The purpose of this research is to identify the production of wingko according to GMP perspective and the quality of Wingko based on SNI-01-4311-1996, and to make GMP plan for the home industries. This is an observational research. Based on the data, this research is observational descriptive, while according to the period of the research, this is a crossectional research. The sample for this research is Wingko, the owner and the employees whoever involve in the production. The variable in this research consists of staple, employees health, production tools, location and facilitations, E.coli and sweetener (saccharin and cyclamate), and last but not least the sensory aspects. The research shows the good categorized variables are: staple, production tools, health employees sensory aspects. Meanwhile the adequate categorized are: location and facilitations. The result of laboratory test over E.coli and sweetener, shows that these are qualifi ed based on SNI 01-4311-1996 about Wingko’s grade standard. The variables that don’t meet the qualifi cations of (GMP), are suggested to be improved to make better results and more qualified, while the qualifi ed variables based on GMP are expected to keep their good conditions.Keywords: Wingko, Food Grade Quality, GMP
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Lechanteur, Chantal, Alexandra Briquet, Virginie Bettonville, Etienne Baudoux, and Yves Beguin. "MSC Manufacturing for Academic Clinical Trials: From a Clinical-Grade to a Full GMP-Compliant Process." Cells 10, no. 6 (May 26, 2021): 1320. http://dx.doi.org/10.3390/cells10061320.
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Following European regulation 1394/2007, mesenchymal stromal cell (MSCs) have become an advanced therapy medicinal product (ATMP) that must be produced following the good manufacturing practice (GMP) standards. We describe the upgrade of our existing clinical-grade MSC manufacturing process to obtain GMP certification. Staff organization, premises/equipment qualification and monitoring, raw materials management, starting materials, technical manufacturing processes, quality controls, and the release, thawing and infusion were substantially reorganized. Numerous studies have been carried out to validate cultures and demonstrate the short-term stability of fresh or thawed products, as well their stability during long-term storage. Detailed results of media simulation tests, validation runs and early MSC batches are presented. We also report the validation of a new variant of the process aiming to prepare fresh MSCs for the treatment of specific lesions of Crohn’s disease by local injection. In conclusion, we have successfully ensured the adaptation of our clinical-grade MSC production process to the GMP requirements. The GMP manufacturing of MSC products is feasible in the academic setting for a limited number of batches with a significant cost increase, but moving to large-scale production necessary for phase III trials would require the involvement of industrial partners.
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Kierkels, Guido J. J., Trudy Straetemans, Moniek A. de Witte, and Jürgen Kuball. "The next step toward GMP-grade production of engineered immune cells." OncoImmunology 5, no. 2 (August 27, 2015): e1076608. http://dx.doi.org/10.1080/2162402x.2015.1076608.
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Heshusius, Steven, Esther Heideveld, Patrick Burger, Marijke Thiel-Valkhof, Erica Sellink, Eszter Varga, Elina Ovchynnikova, et al. "Large-scale in vitro production of red blood cells from human peripheral blood mononuclear cells." Blood Advances 3, no. 21 (November 4, 2019): 3337–50. http://dx.doi.org/10.1182/bloodadvances.2019000689.
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Key Points This article provides a defined GMP-grade medium and erythroid culture protocol, resulting in >90% enucleated RBC. This article provides a high-resolution database of RNA expression dynamics at daily intervals during terminal erythroid differentiation.
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Aussel, Clotilde, Elodie Busson, Helene Vantomme, Juliette Peltzer, and Christophe Martinaud. "Quality assessment of a serum and xenofree medium for the expansion of human GMP-grade mesenchymal stromal cells." PeerJ 10 (May 30, 2022): e13391. http://dx.doi.org/10.7717/peerj.13391.
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Background Cell-based therapies are emerging as a viable modality to treat challenging diseases, resulting in an increasing demand for their large-scale, high-quality production. Production facilities face the issue of batch-to-batch consistency while producing a safe and efficient cell-based product. Controlling culture conditions and particularly media composition is a key factor of success in this challenge. Serum and Xeno-Free Media (SXFM) represent an interesting option to achieve this goal. By reducing batch to batch variability, they increase Good Manufacturing Practices (GMP)-compliance and safety regarding xenogenic transmission, as compared to fetal bovine serum (FBS) supplemented-media or human platelet lysate supplemented medium. Methods In this study, the isolation, expansion and characteristics including the anti-inflammatory function of human mesenchymal stromal cells (MSC) are compared after culture in MEMα supplemented with human Concentrate Platelet Lysate (hCPL, reference medium) or in MSC-Brew GMP Medium. The latter is a GMP SXFM manufactured in bags under strictly controlled conditions in volumes suitable for expansion to a clinical scale and does not require neither pre-coating of the cell culture units nor the addition of blood derivatives at the isolation step. Results We showed that MSC derived from human bone-marrow and adipose tissue can be successfully isolated and expanded in this SXFM. Number and size of Colony-Forming Unit fibroblast (CFU-F) is increased compared to cells cultivated in hCPL medium. All cells retained a CD90+, CD73+, CD105+, HLADR−, CD34−, CD45− phenotype. Furthermore, the osteogenic and adipocyte potentials as well as the anti-inflammatory activity were comparable between culture conditions. All cells reached the release criteria established in our production facility to treat inflammatory pathologies. Conclusions The use of MSC-Brew GMP Medium can therefore be considered for clinical bioprocesses as a safe and efficient substitute for hCPL media.
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Chauvierre, Cédric, Rachida Aid-Launais, Joël Aerts, Frédéric Chaubet, Murielle Maire, Lucas Chollet, Lydia Rolland, et al. "Pharmaceutical Development and Safety Evaluation of a GMP-Grade Fucoidan for Molecular Diagnosis of Cardiovascular Diseases." Marine Drugs 17, no. 12 (December 12, 2019): 699. http://dx.doi.org/10.3390/md17120699.
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The adhesion molecule P-selectin is present on the cell surface of both activated endothelium and activated platelets. The present study describes the pharmaceutical development, safety evaluation, and preclinical efficacy of a micro-dosed radiotracer. The macromolecular nanoscale assembly consisted of a natural compound made of a sulfated fucose-rich polysaccharides (fucoidan) and a radionuclide (technetium-99m) for the detection of P-selectin expression in cardiovascular diseases. After extraction and fractionation from brown seaweeds, the good manufacturing practice (GMP) production of a low molecular weight (LMW) fucoidan of 7 kDa was achieved and full physicochemical characterization was performed. The regulatory toxicology study in rats of the GMP batch of LMW fucoidan revealed no adverse effects up to 400 μg/kg (×500 higher than the expected human dose) and pseudoallergy was not seen as well. In a myocardial ischemia-reperfusion model in rats, the GMP-grade LMW fucoidan labeled with technetium-99m detected P-selectin upregulation in vivo. The present study supports the potential of using 99mTc-fucoidan as an imaging agent to detect activated endothelium in humans.
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Rusconi, Giulio, Giuseppe Cusumano, Luca Mariotta, Reto Canevascini, Mauro Gola, Rosalba Gornati, and Gianni Soldati. "Upgrading Monocytes Therapy for Critical Limb Ischemia Patient Treatment: Pre-Clinical and GMP-Validation Aspects." International Journal of Molecular Sciences 23, no. 20 (October 21, 2022): 12669. http://dx.doi.org/10.3390/ijms232012669.
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Advanced cell therapy medicinal products (ATMP) are at the forefront of a new range of biopharmaceuticals. The use of ATMP has evolved and increased in the last decades, representing a new approach to treating diseases that are not effectively managed with conventional treatments. The standard worldwide recognized for drug production is the Good Manufacturing Practices (GMP), widely used in the pharma production of synthesized drugs but applying also to ATMP. GMP guidelines are worldwide recognized standards to manufacture medicinal products to guarantee high quality, safety, and efficacy. In this report, we describe the pre-clinical and the GMP upgrade of peripheral blood mononuclear cell (PBMC) preparation, starting from peripheral blood and ending up with a GMP-grade clinical product ready to be used in patients with critical limb ischemia (CLI). We also evaluated production in hypoxic conditions to increase PBMC functional activity and angiogenic potential. Furthermore, we extensively analyzed the storage and transport conditions of the final product as required by the regulatory body for ATMPs. Altogether, results suggest that the whole manufacturing process can be performed for clinical application. Peripheral blood collected by a physician should be transported at room temperature, and PBMCs should be isolated in a clean room within 8 h of venipuncture. Frozen cells can be stored in nitrogen vapors and thawed for up to 12 months. PBMCs resuspended in 5% human albumin solution should be stored and transported at 4 °C before injection in patients within 24 h to thawing. Hypoxic conditioning of PBMCs should be implemented for clinical application, as it showed a significant enhancement of PBMC functional activity, in particular with increased adhesion, migration, and oxidative stress resistance. We demonstrated the feasibility and the quality of a GMP-enriched suspension of monocytes as an ATMP, tested in a clean room facility for all aspects related to production in respect of all the GMP criteria that allow its use as an ATMP. We think that these results could ease the way to the clinical application of ATMPs.
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Wagner, Michael, Johan G. Doverfjord, Joachim Tillner, Gunnar Antoni, Torsten Haack, Martin Bossart, Iina Laitinen, et al. "Automated GMP-Compliant Production of [68Ga]Ga-DO3A-Tuna-2 for PET Microdosing Studies of the Glucagon Receptor in Humans." Pharmaceuticals 13, no. 8 (July 31, 2020): 176. http://dx.doi.org/10.3390/ph13080176.
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Introduction: [68Ga]Ga-DO3A-VS-Cys40-Tuna-2 (previously published as [68Ga]Ga-DO3A-VS-Cys40-S01-GCG) has shown high-affinity specific binding to the glucagon receptor (GCGR) in vitro and in vivo in rats and non-human primates in our previous studies, confirming the suitability of the tracer for drug development applications in humans. The manufacturing process of [68Ga]Ga-DO3A-VS-Cys40-Tuna-2 was automated for clinical use to meet the radiation safety and good manufacturing practice (GMP) requirements. Methods: The automated synthesis platform (Modular-Lab PharmTrace, Eckert & Ziegler, Eurotope, Germany), disposable cassettes for 68Ga-labeling, and pharmaceutical-grade 68Ge/68Ga generator (GalliaPharm®) used in the study were purchased from Eckert & Ziegler. The parameters such as time, temperature, precursor concentration, radical scavenger, buffer concentration, and pH, as well as product purification step, were investigated and optimized. Process optimization was conducted with regard to product quality and quantity, as well as process reproducibility. The active pharmaceutical ingredient starting material DO3A-VS-Cys40-Tuna-2 (GMP-grade) was provided by Sanofi Aventis. Results: The reproducible and GMP-compliant automated production of [68Ga]Ga-DO3A-VS-Cys40-Tuna-2 with on-line documentation was developed. The non-decay-corrected radiochemical yield was 45.2 ± 2.5% (n = 3, process validation) at the end of the synthesis with a labeling synthesis duration of 38 min and a quality controlincluding release procedure of 20 min. The radiochemical purity of the product was 98.9 ± 0.6% (n = 17) with the total amount of the peptide in the preparation of 48 ± 2 µg (n = 3, process validation). Radionuclidic purity, sterility, endotoxin content, residual solvent content, and sterile filter integrity tests met the acceptance criteria. The product was stable at ambient temperature for at least 2 h. Conclusion: The fully automated GMP-compliant manufacturing process was developed and thoroughly validated. The resulting [68Ga]Ga-DO3A-VS-Cys40-Tuna-2 was used in a clinical study for accurate quantification of GCGR occupancy by a dual anti-diabetic drug in vivo in humans.
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Malthufah, Yeda Rachma Ayu, and Kusuma Scorpia Lestari. "Assisting Food Household Industries in Implementation of Good Manufacturing Practice (GMP) on Frozen Meat Kebab in Sidoarjo District." Media Gizi Kesmas 12, no. 2 (November 30, 2023): 878–85. http://dx.doi.org/10.20473/mgk.v12i2.2023.878-885.
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Background: Good Manufacturing Practice (GMP) is a reference that explains the procedures to produce processed food so that it is of high quality, safe, and suitable for public consumption. The Food Safety Facilitator's role is to help register the processed products of SME (Small Medium Enterprise) X to obtain an NIE( Nomor Izin Edar) number. Objectives: The SME assistance aims to improve the implementation of GMP so that it can meet the GMP level that meets the requirements of the Regulation of the Head of the Food and Drug Administration on Guidelines for Inspection of Processed Food Production Facilities. Methods: Initial observations were made with a form based on the standards of the Food and Drug Administration to determine the score and value of SME X, then mentoring and training were carried out to correct GMP non-conformities. Results: The first observation of SME X showed that there were aspects that did not comply with the GMP requirements. With several non-conformities obtained, SME X received a grade of D or very poor, so it needs improvement to get an NIE number from BPOM. The results of the final inspection indicated an improvement in the score from D (very poor) to B (good) so that SME X could obtain the NIE number. Conclusions: Assistance and training to SMEs contributed to the improvement of SMEs' GMP implementation scores so that SME X was able to obtain a distribution license number for its food products
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Kropp, Martina, Nina Harmening, Thais Bascuas, Sandra Johnen, Eline De Clerck, Verónica Fernández, Mattia Ronchetti, et al. "GMP-Grade Manufacturing and Quality Control of a Non-Virally Engineered Advanced Therapy Medicinal Product for Personalized Treatment of Age-Related Macular Degeneration." Biomedicines 10, no. 11 (November 1, 2022): 2777. http://dx.doi.org/10.3390/biomedicines10112777.
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The introduction of new therapeutics requires validation of Good Manufacturing Practice (GMP)-grade manufacturing including suitable quality controls. This is challenging for Advanced Therapy Medicinal Products (ATMP) with personalized batches. We have developed a person-alized, cell-based gene therapy to treat age-related macular degeneration and established a vali-dation strategy of the GMP-grade manufacture for the ATMP; manufacturing and quality control were challenging due to a low cell number, batch-to-batch variability and short production duration. Instead of patient iris pigment epithelial cells, human donor tissue was used to produce the transfected cell product (“tIPE”). We implemented an extended validation of 104 tIPE productions. Procedure, operators and devices have been validated and qualified by determining cell number, viability, extracellular DNA, sterility, duration, temperature and volume. Transfected autologous cells were transplanted to rabbits verifying feasibility of the treatment. A container has been engineered to ensure a safe transport from the production to the surgery site. Criteria for successful validation and qualification were based on tIPE’s Critical Quality Attributes and Process Parameters, its manufacture and release criteria. The validated process and qualified operators are essential to bring the ATMP into clinic and offer a general strategy for the transfer to other manufacture centers and personalized ATMPs.
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Nicod, Clémentine, Mathieu Neto Da Rocha, Xavier Roussel, Rafik Haderbache, Rim Trad, Lucie Bouquet, Walid Warda, et al. "Production préclinique de CART cells IL-1RAP “GMP-like” en vue d’un essai Clinique de phase Ib.–Expérience bisontine." Transfusion Clinique et Biologique 28, no. 4 (November 2021): S4. http://dx.doi.org/10.1016/j.tracli.2021.08.018.
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Poorebrahim, Mansour, Solmaz Sadeghi, Elham Fakhr, Mohammad Foad Abazari, Vahdat Poortahmasebi, Asma Kheirollahi, Hassan Askari, et al. "Production of CAR T-cells by GMP-grade lentiviral vectors: latest advances and future prospects." Critical Reviews in Clinical Laboratory Sciences 56, no. 6 (July 17, 2019): 393–419. http://dx.doi.org/10.1080/10408363.2019.1633512.
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Zielhuis, S. W., J. F. W. Nijsen, R. de Roos, G. C. Krijger, P. P. van Rijk, W. E. Hennink, and A. D. van het Schip. "Production of GMP-grade radioactive holmium loaded poly(l-lactic acid) microspheres for clinical application." International Journal of Pharmaceutics 311, no. 1-2 (March 2006): 69–74. http://dx.doi.org/10.1016/j.ijpharm.2005.12.034.
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Shaim, Hila, Mayela Carolina Mendt, Hani Lee, Bijender Kumar, Min Soon Cho, Alejandro Ramirez, Indreshpal Kaur, et al. "Large Scale Manufacturing of GMP Grade Cord Blood Derived Megakaryocytes for Chemotherapy Induced Thrombocytopenia." Blood 144, Supplement 1 (November 5, 2024): 3926. https://doi.org/10.1182/blood-2024-209206.
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Introduction: A reliable and sustainable supply of platelets remains a critical challenge in managing thrombocytopenic patients, with over 2 million platelet units transfused annually in the United States alone. This demand is common in patients undergoing treatments such as chemotherapy or bone marrow transplantation, who frequently require platelet transfusions due to the short life span of platelets. It can be further exacerbated by immune refractoriness to platelet transfusions. Currently, hospitals rely heavily on adult donor apheresis to meet this need; however, the short storage life of platelets and donor availability limit this supply. We thus developed a method of large-scale ex vivo production of good manufacturing practice (GMP) grade platelet and platelet progenitor cells from CD34+ cord blood (CB) cells co-cultured with mesenchymal stem cells (MSCs) derived from CB using hollowfiber flow-based bioreactors. Our approach aims to establish a continuous and renewable source of platelets from the large global cord blood bank inventories. Methods: We developed a dynamic protocol for the large-scale production of expanded megakaryocyte (MKs) and platelets in vitro, employing a coculture system with CB MSCs, the addition of dynamic concentrations of MK differentiation factors, and chemical inhibition of Rho-associated protein kinase (ROCK). Cord blood and CB MSCs were obtained from the MDACC Cord Blood and MSC Banks, respectively. Our manufacturing process consists of three phases: the first phase involves the co-culturing fresh CB-derived CD34+ cells (enriched to >90% using MACS enrichment columns, Milteny Biotec) with monolayers of CB MSCs cells for 3 days, using GMP media supplemented with MK early differentiation cytokines. During the second phase, the expanded CD34+ cells and MK progenitors were cultured using GMP media supplemented with late differentiation factors including high concentrations of thrombopoietin (TPO) for 7 days. At day 10, the cells were transferred to the hollowfiber bioreactor, where they were co-cultured for 9 days with MSCs in GMP media supplemented with MK late differentiation factors and a lower concentration of TPO. In the final phase, the MKs were treated with a ROCK inhibitor for 4 days, to promote further MK maturation and release of platelets. On Day 23, the bioreactor was harvested, and the final product consisting of platelets and expanded MKs were washed, analyzed by flow cytometry and infused into xenogeic NSG mice. Results: After a 23 day culture, our final product contains 113 x 106 ±29.2 megakaryocytes (CD41+CD61+) and 0.4-0.9 x 1011 platelets. The cultured MK were polyploid, and the platelets demonstrated functional equivalence to control human platelets, as confirmed by in vitro aggregation studies. To evaluate the in vivo efficacy and functionality of the product, we transfused a combination of 106 megakaryocytes and 20 x 106 platelets into sublethally irradiated NSG mice. Post-transfusion assessments revealed a significant reduction in the tail vein bleeding time 24 hours later. Moreover, human platelet production was detected in the peripheral blood of the mice up to 2 weeks following the transfusion, suggesting that the administered MKs were actively producing platelets in vivo. Importantly, the product was well-tolerated with no observed toxicity in the mice. Our results highlight the clinical feasibility of this approach. In addition to MSC co-culture and ROCK inhibition, the novelty of this approach includes the transfusion of a product containing both megakaryocytes and platelets, offering a promising strategy for addressing platelet shortages and improving patient outcomes. Conclusions: We standardized a GMP-compliant protocol for the manufacture of megakaryocytes and platelets from CD34+ derived from Cord Blood. Large-scale expansion of functional MKs and platelets can be generated efficiently in the bioreactor system for clinical use. Clinical trials to evaluate this new product are planned.
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Zanten, Jacoba van, Annelies Jorritsma-Smit, Hans Westra, Mirjam Baanstra, Anne de Bruin-Jellema, Derk Allersma, Bahez Gareb, and Rob P. Coppes. "Optimization of the Production Process of Clinical-Grade Human Salivary Gland Organoid-Derived Cell Therapy for the Treatment of Radiation-Induced Xerostomia in Head and Neck Cancer." Pharmaceutics 16, no. 3 (March 21, 2024): 435. http://dx.doi.org/10.3390/pharmaceutics16030435.
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Head and neck cancer is a common cancer worldwide. Radiotherapy has an essential role in the treatment of head and neck cancers. After irradiation, early effects of reduced saliva flow and hampered water secretion are seen, along with cell loss and a decline in amylase production. Currently, there is no curative treatment for radiation-induced hyposalivation/xerostomia. This study aimed to develop and optimize a validated manufacturing process for salivary gland organoid cells containing stem/progenitor cells using salivary gland patient biopsies as a starting material. The manufacturing process should comply with GMP requirements to ensure clinical applicability. A laboratory-scale process was further developed into a good manufacturing practice (GMP) process. Clinical-grade batches complying with set acceptance and stability criteria were manufactured. The results showed that the manufactured salivary gland-derived cells were able to self-renew, differentiate, and show functionality. This study describes the optimization of an innovative and promising novel cell-based therapy.
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Labarriere, N., A. Fortun, A. Bellec, A. Khammari, B. Dreno, S. Saïagh, and F. Lang. "A Full GMP Process to Select and Amplify Epitope-Specific T Lymphocytes for Adoptive Immunotherapy of Metastatic Melanoma." Clinical and Developmental Immunology 2013 (2013): 1–11. http://dx.doi.org/10.1155/2013/932318.
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A number of trials of adoptive transfer of tumor-specific T lymphocytes have been performed in the last 20 years in metastatic melanoma, with increasingly encouraging results as the relevant melanoma antigens were identified and the purity/specificity of injected T cells improved. We have previously described a sorting method of epitope-specific T lymphocytes that uses magnetic beads coated with HLA/peptide complexes and we suggested that this method could be applied to a clinical setting. In the present work, we provide a detailed description of the whole GMP process of sorting and amplification of clinical grade T cells specific for the melanoma antigens Melan-A and MELOE-1. All the reagents used in this process including the sorting reagent were produced in GMP conditions and we document the optimization of the different steps of the process such as peptide stimulation, sorting, and amplification. The optimized procedure, validated in 3 blank runs in a clinical setting, allowed the production of at least 108pure (>90%) Melan-A- and MELOE-1-specific T cells within 28 days starting with 100 mL of blood from metastatic melanoma patients. This GMP process is thus ready to be used in an upcoming phase I/II clinical trial on metastatic melanoma patients.
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Becker, Fabienne, Stefanie Liedtke, Boris Greber, Nils Kriedemann, Ulrich Martin, Robert Zweigerdt, and Gesine Koegler. "Abstract 23 HLA-Homozygous iPSC from Cord Blood for the Generation of Cardiomyocytes for Allogeneic Cell Therapy to the Heart." Stem Cells Translational Medicine 12, Supplement_1 (September 1, 2023): S25. http://dx.doi.org/10.1093/stcltm/szad047.024.
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Abstract Introduction Based on a previous grant on HLA-homozygous (HLAh) iPS-GMP (and ATMP conform) generation from allogeneic licensed cord blood (CB) units, the Düsseldorf CBB could provide the most frequent HLA-types for reprogramming (Liedtke et al. 2020). These HLAh iPSC provide the basis for the generation of cardiomyocytes from iPSC. Patients with acute heart failure or cardiomyopathies have limited options of treatment. iPSC technology allows for de novo generation of functional heart muscle in vitro, enabling the implementation of new therapy concepts for heart regeneration. Objectives The GMP-grade iPSC-lines are the backbone for the granted EU project (HEAL (101056712) on iPSC-cardiomyocyte aggregate manufacturing for allogeneic cell therapy. Besides GMP-compliant up-scaled cell production of cardiomyocytes, the project is designed to overcome many of the scientific, safety and regulatory hurdles necessary to initiate a first-in-man clinical HLAh-based heart regeneration study. Methods After CD34+ selection/short expansion of CD34, cells were reprogrammed by GMP-conform plasmids and stored in a master cell bank with most frequent HLA-haplotypes. All iPSC lines were tested extensively for genetic stability, mycoplasma, virus markers, endotoxins and microorganisms. The differentiation into cardiomyocytes was performed in correspondence to the Wnt modulation, i.e. first activating Wnt pathway and then inhibiting the pathway with the addition of small molecules (Halloin et al. 2019). For the up-scaled production, a 3D-cardiomyocyte aggregate culture in large stirred tank bioreactors (STBR) was applied. Cryopreservation using controlled rate freezing was adapted to tissue freezing. Results Selection of the most frequent homozygous haplotypes facilitated the production of HLAh iPSC lines for differentiation into cardiomyocytes. Efficient progress had been made for the improvement of STBR platform for sufficient production of cardiomyocytes, enabling the differentiation with a purity of up to 95%, measured via cardiac troponin T (cTnT). Discussion Although high yields of cardiomyocytes in a GMP-compliant process is getting more achievable by using STBRs, the safety and regulatory hurdles are still a major problem. For safety testing, toxicology, tumorigenicity assays will be performed in mice and in pig models. This will enable the prediction of arrhythmia-related risks. For a safe distribution of the finally released cell product optimization of cryopreservation of cardiomyocytes aggregates will be performed. Disclaimer: Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Health and Digital Executive Agency (HADEA). Neither the European Union nor the granting authority can be held responsible for them.
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Menard, Cedric, Luciano Pacelli, Giulio Bassi, Joelle Dulong, Isabelle Bezier, Jasmina Zanoncello, Francesco Bifari, et al. "Quality Controls Of Immune Regulatory Properties Of Ex-Vivo, GMP-Grade Expanded Mesenchymal Stromal Cells For Clinical Use." Blood 122, no. 21 (November 15, 2013): 5413. http://dx.doi.org/10.1182/blood.v122.21.5413.5413.
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Abstract Aim of CASCADE is to standardize GMP-grade production and clinical use of Mesenchymal Stromal Cells (MSC). Immunological Unit is aimed at setting up and validating a standardized panel of functional assays to fully characterize the immunomodulatory properties of MSC obtained from bone marrow and adipose tissue through different GMP-grade expansion protocols (platelet lysate- vs. fetal calf serum). Immune cells were isolated using indirect immunomagnetic depletion (purity >96%). MSC were expanded in the same medium used for production and harvested at 70% confluence. Primed MSC (pMSC) were obtained after 48h-treatment with rh-IFNγ and rh-TNFα. MSC or pMSC were cocultured with T, B, NK cells for 4 or 6 days, and proliferation was evaluated by CFDA-SE dilution. T cells were stimulated with αCD3 + αCD28 antibodies; B cells were activated with CD40L, its enhancer, IL-2, CpG 2006, and anti-IgM/IgA/IgG; NK cells were activated with 100 U/ml rhIL-2. Cocultures were performed also with specific molecule inhibitors: L-1MT (IDO), snPP (HO-1), NS-398 (COX2), L-NMMA (iNOS) and anti-IFNγ antibody. For MSC immunogenicity assay, allogeneic T cell proliferation was evaluated at day 5 of culture; in addition, NK cells were activated for 2 days with rh-IL2, and MSC and pMSC were used as target cells. Inflammatory milieu significantly upregulated MHC class I and II, CD54, CD106, CD40, CD274, CD112, CD155 expression, and downregulated NKG2D ligands and mesenchymal markers (CD73, CD90, CD105). AT-derived MSC expressed less MHC class II, CD200 and CD106 molecules than BM-MSC. MSC coculture inhibited T and NK cell proliferation without inducing apoptosis, and this effect was greater in presence of primed MSC. Only primed MSC were capable of suppressing B cell proliferation. MSC inhibited apoptosis of resting T, B, and NK cells, while inflammatory priming increased their pro-survival activity. Activation of IDO and HO-1 was the main mechanism involved in MSC immune modulation. MSC never promoted allogeneic T cell proliferation; by contrast, IL-2-activated NK cells could efficiently recognize and kill allogenic unprimed MSC, while primed MSC became insensitive to NK cells. Some differences were observed depending on the origin and culture conditions of clinical-grade MSC. All the experimental protocols to assess MSC inhibitory effects on immune effector cells have been standardized and will be applied for the release of GMP-grade MSC produced inside the CASCADE Consortium. Disclosures: No relevant conflicts of interest to declare.
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Lara Chica, Maribel, María De La Rosa-Garrido, Blanca Arribas-Arribas, Miguel-Ángel Montiel-Aguilera, Raquel Muñoz-García, María Bermejo-González, Esteban Márquez-Pérez, et al. "Validation of GMP Lentiviral Vector Production for CAR T-Cell Therapy in the Spanish Public Health System: Development of New QC Required By Aemps (Spanish FDA)." Blood 144, Supplement 1 (November 5, 2024): 7257. https://doi.org/10.1182/blood-2024-207219.
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The production of Lentiviral Vectors (LV) for academic CAR T-cell therapies under Good Manufacturing Practices (GMP) is the main bottleneck within the production process. We have developed a GMP facility (ViPro-IBIS) according to the AEMPS (“Spanish Drug and Health Products Agency”), including: - Facilities and equipment: grade qualifications (main production room and changing rooms), equipment qualifications, validation of the cleanliness of the qualified facilities as well as validation of the entry of materials into them. - Production processes: validation of Master Cell Bank (MCB) and Working Cell Banks (WCB) production of packaging cells (HEK293T “Lenti-X”), validation of LV production and validation of media fill (MF) processes (3 consecutive successful productions in all cases). All the mentioned validations have been successfully completed, complying with all the requirements and quality controls (QC) requested by the AEMPS. Of these, some have been requested for the first time (highlighted in italics), which has required an extra effort of fine-tuning: MCB and WCB production: microbiological and particle process monitoring; cell viability ≥ 70% and ≥ 170x106 total live cells achieved (cell viability ≥ 94% in all cases, total live cells achieved: MCB = 352,5x106, WCB1 = 170x106, WCB2 = 267,1x106, WCB3 = 310,5x106); sterility; absence of mycoplasma; absence of endotoxins; cell identity by DNA Fingerprint (Short Tamdem Repeat (STR), only for MCB); analysis of genetic stability; cell duplication calculation (only for WCB); analysis of the cytopathic effect with VERO, MRC-5 and RD cell lines for at least 14 days; analysis of human adventitious viruses by PCR and RT-PCR; analysis of porcine viruses by PCR and RT-PCR (only for MCB); control of endogenous retroviruses by detection of reverse transcriptase activity (only for MCB). LV production: microbiological and particle process monitoring; cell viability ≥ 70% (≥ 90% in all cases); sterility; absence of mycoplasma; absence of endotoxins; functional LV titer measured by FACS ≥1x107 TU/mL (LV1 = 3.07x108 TU/mL, LV2 = 9.95x107 TU/mL, LV3 = 1.66x108 TU/mL); analysis of DNA (by digital PCR) and protein (by WB) residues from packaging cells and residual DNA from plasmids (by digital PCR) used in the production process. All LV batches were packed in bags with 250x106+10% lentiviral particles diluted in TexMACS™ GMP Medium (Miltenyi), in accordance with the specifications. Moreover, the LV stability was set at at least one year. Currently, we have submitted the application for AEMPS inspection, which we expect to occur during the second semester of 2024. Thus, GMP LV production at ViPro-IBIS represents a robust platform within the Spanish public health system for CAR T-cell therapy clinical trials, allowing to provide LV in a more rapid and affordable way for the trials approved by the AEMPS in the region/country (including ours: SPCD19M-CAR for lymphoma and CARTemis-1 (BCMA CAR) for myeloma). Our platform may serve as a model for improvement for CAR T-cell treatments for the patients.
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Van der Loo, Johannes C. M., William Swaney, Diana Nordling, Axel Schambach, Christopher Baum, David A. Williams, Lilith Reeves, and Punam Malik. "Production of High Titer cGMP-Grade SIN Gamma-Retroviral Vectors by Transfection in a Closed System Bioreactor." Blood 112, no. 11 (November 16, 2008): 3539. http://dx.doi.org/10.1182/blood.v112.11.3539.3539.
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Abstract The need for gamma-retroviral vectors with self-inactivating (SIN) long terminal repeats for clinical trials has prompted a shift in the method with which large scale GMP-grade vectors are produced, from the use of stable producer lines to transient transfection-based techniques. The main challenge of instituting this methodology was to develop SIN retrovirus vectors that produced high amounts of genomic vector RNA in packaging cells, and to design scalable processes for closed system culture, transfection and virus harvest. Using improved expression plasmids, the Vector Production Facility, an academic GMP manufacturing laboratory that is part of the Translational Cores at Cincinnati Children’s Hospital, has developed such a method based on the Wave Bioreactor® production platform. In brief, cells from a certified 293T master cell bank are expanded, mixed with transfection reagents, and pumped into a 2, 10 or 20 Liter Wave Cell Bag containing FibraCel® discs. Cells are cultured in DMEM with GlutaMax® and 10% FBS at 37°C, 5% CO2 at a rocking speed of 22 rpm and 6° angle. At 16–20 hrs post-transfection, the media is changed; virus is harvested at 12-hour intervals, filtered through a leukocyte reduction filter, aliquoted into Cryocyte freezing containers, and frozen at or below −70°C. Several processing parameters, including the confluency of cells harvested prior to transfection, the timing of transfection, the amount of plasmid DNA, exposure of cells to PBS/TrypLESelect, and the timing of the media change post-transfection affected vector titer. Mixing cells with plasmid and transfection mixture prior to seeding onto FibraCel, as compared to transfecting cells 1-day post-seeding (as is standard when using tissue culture plastic) increased the titer from 104 to 4 × 105 IU/mL. Similarly, increasing the amount of plasmid DNA per mL from 4.6 to 9.2 μg doubled the titer in the Wave, while it reduced titer by 20–40% in tissue culture flasks (Fig. 1). Using an optimized protocol, six cGMP-grade SIN gamma-retroviral vectors have now been produced in support of the FDA’s National Toxicology Program (NTP), with unconcentrated vector titers ranging from 1 × 106 to as high as 4 × 107 IU/mL. Using similar processing, we have produced a large scale SIN gamma-retroviral vector (GALV pseudotyped) for an international X-linked SCID trial with average unconcentrated titers of 106 IU/mL in all viral harvests. In summary, the process developed at the Cincinnati Children’s Hospital Vector Production Facility allows for large scale closed-system production of high-titer retroviral vectors for clinical trials using transient transfection. Figure Figure
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Montemurro, Tiziana, Cristiana Lavazza, Elisa Montelatici, Silvia Budelli, Salvatore La Rosa, Mario Barilani, Cecilia Mei, et al. "Off-the-Shelf Cord-Blood Mesenchymal Stromal Cells: Production, Quality Control, and Clinical Use." Cells 13, no. 12 (June 19, 2024): 1066. http://dx.doi.org/10.3390/cells13121066.
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Background: Recently, mesenchymal stromal cells (MSCs) have gained recognition for their clinical utility in transplantation to induce tolerance and to improve/replace pharmacological immunosuppression. Cord blood (CB)-derived MSCs are particularly attractive for their immunological naivety and peculiar anti-inflammatory and anti-apoptotic properties. Objectives: The objective of this study was to obtain an inventory of CB MSCs able to support large-scale advanced therapy medicinal product (ATMP)-based clinical trials. Study design: We isolated MSCs by plastic adherence in a GMP-compliant culture system. We established a well-characterized master cell bank and expanded a working cell bank to generate batches of finished MSC(CB) products certified for clinical use. The MSC(CB) produced by our facility was used in approved clinical trials or for therapeutic use, following single-patient authorization as an immune-suppressant agent. Results: We show the feasibility of a well-defined MSC manufacturing process and describe the main indications for which the MSCs were employed. We delve into a regulatory framework governing advanced therapy medicinal products (ATMPs), emphasizing the need of stringent quality control and safety assessments. From March 2012 to June 2023, 263 of our Good Manufacturing Practice (GMP)-certified MSC(CB) preparations were administered as ATMPs in 40 subjects affected by Graft-vs.-Host Disease, nephrotic syndrome, or bronco-pulmonary dysplasia of the newborn. There was no infusion-related adverse event. No patient experienced any grade toxicity. Encouraging preliminary outcome results were reported. Clinical response was registered in the majority of patients treated under therapeutic use authorization. Conclusions: Our 10 years of experience with MSC(CB) described here provides valuable insights into the use of this innovative cell product in immune-mediated diseases.
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Okafor, Ngozi. "Application of Online Pedagogy to Enhance Undergraduates’ Learning Outcomes in Chemistry." Science Education International 33, no. 3 (September 1, 2022): 284–90. http://dx.doi.org/10.33828/sei.v33.i3.3.
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The low Cumulative Grade Point Average of most chemistry undergraduates results in their low achievement and motivation which poses a threat to the production of future chemistry teachers. The diversity of this problem has resulted in several suggestions concerning the application of Google Meet strategy to determine its efficacy in ameliorating this problem in the Nigerian educational setting. Two null hypotheses guided the research. This study was an ex-post facto and quasi-experimental designs that involved 125 chemistry undergraduates. Three research instruments were used in data collection. Data were analyzed using mean and analysis of variance. Results showed that undergraduates exposed to Google Meet Pedagogy (GMP) had greater achievement than those exposed to Classroom Teacher Approach (CTA). However, undergraduates exposed to CTA had better motivation in chemistry than those exposed to GMP. The finding showed a significant influence of course types on undergraduates’ achievement in chemistry. There was no significant influence of course types on undergraduates’ motivation. The study concludes that undergraduates should be provided with adequate online facilities to be skillful and knowledgeable. It suggests that university lecturers should be sponsored for professional development on the application of online pedagogies to enhance undergraduates’ achievement and motivation in chemistry.
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Winkler, Julia, Michael Mach, Juergen Zingsem, Volker Weisbach, Andreas Mackensen, and Thomas H. Winkler. "GMP-Grade Generation of B-Lymphocytes for Adoptive Immunotherapy in Patients After Allogeneic Stem Cell Transplantation." Blood 120, no. 21 (November 16, 2012): 4352. http://dx.doi.org/10.1182/blood.v120.21.4352.4352.
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Abstract Abstract 4352 Background and objectives: We have recently shown that memory B-lymphocytes from murine CMV immune donor animals adoptively transferred into immunodeficient mice were highly effective in protecting from a viral infection indicating a therapeutic potential of virus specific memory B-cells. These preclinical data provided evidence that a cell-based strategy supporting the humoral immune response might be effective in a clinical setting of post-HSCT immunodeficiency (Klenovsek et al., 2007, Blood 110: 3472–9). As adoptive transfer of B-cells has not been used before in a clinical setting, it is necessary to establish a technology for the generation of GMP-grade B-cell products. Methods: Starting from the leukapheresis of healthy donors, B-cells were purified by two different separation strategies using GMP-grade microbeads and the CliniMACS∧TM device. A one-step protocol was used for positive enrichment of B-lymphocytes with anti-CD19 microbeads. In a two-step enrichment protocol, first T-lymphocytes were depleted by anti-CD3 microbeads and the remaining fraction was positively selected by anti-CD19 microbeads. Results: The leukapheresis contained a mean of 9.0×10∧8 CD19-positive B-cells (4.5–12.4 ×10∧8). After the one-step positive purification strategy a mean purity of CD20∧+ B-lymphocytes of 78.1% with a recovery of 32–41% was obtained. With the two-step T-cell depletion/B-cell enrichment protocol we achieved a mean purity of 96.4 % (93.4–97.8%) with a slightly lower recovery of 14–37%. The absolute B-cell numbers obtained in the product were 1.3 to 4.0 ×10∧8 and 1.7 to 2.6 ×10∧8 for the one-step positive enrichment and the two-step protocol, respectively. Importantly, the absolute number of T-cells was lower in cell products after the two-step protocol (0.1 to 0.9 ×10∧6 T-cells) as compared to the one-step positive CD19-enrichment (1.6 to 3.4 ×10∧6 T-cells). Assuming a patient with 70 kg body weight, the B-cell products obtained after the combined CD3-depletion and CD19-enrichment contained less then 4×10∧4 T-lymphocytes/kg bodyweight, which is a critical threshold number of T-cells in haploidentical HSCT. The B-cell products showed antibody production after in vitro stimulation in a limiting dilution assay and showed excellent viability after cryopreservation. Conclusions: A GMP-grade B-cell product can be obtained with high purity and very low T-cell contamination using the two-step enrichment protocol based on CliniMACS∧TM technology. (Supported by BayImmuNet) Disclosures: No relevant conflicts of interest to declare.
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Douay, Luc. "In Vitro Production of Erythrocytes." Blood 120, no. 21 (November 16, 2012): SCI—39—SCI—39. http://dx.doi.org/10.1182/blood.v120.21.sci-39.sci-39.
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Abstract Abstract SCI-39 The generation of red blood cells (RBCs) in vitro using biotechnologies could represent an interesting alternative to classical transfusion products, in that it would combine adequate supplies with the specific production of blood products of a particular phenotype and the reduction of infection risks. This presentation will review how it is now possible to obtain in vitro complete maturation of the erythroid line to the stage of enucleation, starting from hematopoietic stem cells (HSCs) from peripheral blood, bone marrow or umbilical cord blood, or embryonic stem cells or adult pluripotent stem cells (induced pluripotent stem cells, iPSCs). This presentation will discuss how the functionality of cultured human RBCs (cRBCs) is settled in terms of deformability, hemoglobin maturation, oxygen carrying capacity, enzyme content, and terminal maturation from the reticulocyte stage to mature RBC after infusion into the NOD/SCID mouse model. The clinical feasibility of this concept has recently been demonstrated by reporting that cRBCs generated in vitro from peripheral HSCs under GMP conditions encounter in vivo the conditions required for their maturation and that they persist in the circulation for several weeks in humans. These data have established the proof of principle for transfusion of in vitro-generated RBCs and the pathway toward new developments in transfusion medicine. The most proliferative source of stem cells for generating cRBCs is cord blood, but it is limited in terms of HSCs and is dependent on donations. Pluripotent stem cell technology represents a potentially unlimited source of RBCs and opens the door to the development of a new generation of allogeneic transfusion products. Because iPSCs can be selected for a phenotype of interest, they are obviously the best candidate for organizing complementary sources of RBCs for transfusion. It is established that only three human iPSC clones would have been sufficient to match more than 99 percent of the patients in need of RBC transfusions. As a whole, a very limited number of RBC clones would provide for the needs of most alloimmunized patients and those with a rare blood group. Generating cRBCs from iPSCs has been done but needs to be optimized to lead to a clinical application in blood transfusion. Several crucial points remain to be resolved, notably, the choice of the initial cell type, the method of reprogramming (i.e., to ensure the safety of the iPSCs and to ensure their clinical grade), the optimization of the erythrocyte differentiation, and the definition of GMP conditions for industrial production. Assuming that in vitro large-scale cultured RBC production efficiently operates in the near future, this presentation will highlight the potential applications for alloimmunized patients and those with a rare blood group. Disclosures: No relevant conflicts of interest to declare.
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Qi, Yiyao, Xicheng Wang, Zhihui Bai, Ying Xu, Tingting Lu, Hanyu Zhu, Shoumei Zhang, et al. "Enhancement of the function of mesenchymal stem cells by using a GMP-grade three-dimensional hypoxic large-scale production system." Heliyon 10, no. 10 (May 2024): e30968. http://dx.doi.org/10.1016/j.heliyon.2024.e30968.
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Grau-Vorster, Marta, Luciano Rodríguez, Anna del Mazo-Barbara, Clémentine Mirabel, Margarita Blanco, Margarita Codinach, Susana G. Gómez, Sergi Querol, Joan García-López, and Joaquim Vives. "Compliance with Good Manufacturing Practice in the Assessment of Immunomodulation Potential of Clinical Grade Multipotent Mesenchymal Stromal Cells Derived from Wharton’s Jelly." Cells 8, no. 5 (May 21, 2019): 484. http://dx.doi.org/10.3390/cells8050484.
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Background: The selection of assays suitable for testing the potency of clinical grade multipotent mesenchymal stromal cell (MSC)-based products and its interpretation is a challenge for both developers and regulators. Here, we present a bioprocess design for the production of Wharton’s jelly (WJ)-derived MSCs and a validated immunopotency assay approved by the competent regulatory authority for batch release together with the study of failure modes in the bioprocess with potential impact on critical quality attributes (CQA) of the final product. Methods: The lymphocyte proliferation assay was used for determining the immunopotency of WJ-MSCs and validated under good manufacturing practices (GMP). Moreover, failure mode effects analysis (FMEA) was used to identify and quantify the potential impact of different unexpected situations on the CQA. Results: A production process based on a two-tiered cell banking strategy resulted in batches with sufficient numbers of cells for clinical use in compliance with approved specifications including MSC identity (expressing CD73, CD90, CD105, but not CD31, CD45, or HLA-DR). Remarkably, all batches showed high capacity to inhibit the proliferation of activated lymphocytes. Moreover, implementation of risk management tools led to an in-depth understanding of the manufacturing process as well as the identification of weak points to be reinforced. Conclusions: The bioprocess design showed here together with detailed risk management and the use of a robust method for immunomodulation potency testing allowed for the robust production of clinical-grade WJ-MSCs under pharmaceutical standards.
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Molostova, Olga, Larisa Shelikhova, Dina Schneider, Rimma Khismatullina, Yakov Muzalevsky, Alexey Kazachenok, Liane Preussner, et al. "Local Manufacture of CD19 CAR-T Cells Using an Automated Closed-System: Robust Manufacturing and High Clinical Efficacy with Low Toxicities." Blood 134, Supplement_1 (November 13, 2019): 2625. http://dx.doi.org/10.1182/blood-2019-130370.
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Introduction CD19 CAR-T cell products were recently approved as therapy for B-lineage malignancies. We initiated an IIT trial where manufacture of CAR-T cells was performed locally using a unique CD19 CAR with potent anti-leukemic effects. Patients and methods A total of 37 pts with relapsed/refractory B-acute lymphoblastic leukemia (12 female, 25 male, median age 10 y) were screened, 27 pts were enrolled for a trial, 10 were eligible for compassionate use of CD19 CAR-T cell therapy. Sixteen patients had relapsed B-ALL after haploidentical HSCT, 19 pts refractory relapse, 2 induction failure, 13 patients had previous blinatumomab infusion. Eighteen patients had >20% blast cells, median bone marrow leukemia burden for patients with full blown disease was 89%, 19 pts had minimal residual disease (MRD) >0.1% in BM, 3 had skeletal involvement with multiple mass lesions, one had CNS involvement. The CliniMACS Prodigy T cell transduction (TCT) process was used to produce CD19 CAR-T cells. The automated production included CD4/CD8 selection, CD3/CD28 stimulation with MACS GMP T Cell TransAct and transduced with lentiviral vector expressing the CD19CAR gene (second generation CD19.4-1BB zeta with alternate transmembrane domain derived from the TNF superfamily) (Lentigen, Miltenyi Biotec company). T cells were expansion over 10 days in the presence of serum-free TexMACS GMP Medium supplemented with MACS GMP IL-7 and IL-15. Final product was administered without cryopreservation to the patients after fludarabine/cyclophosphamide preconditioning. All patients received prophylactic tocilizumab at 8mg/kg before CAR-T cell infusion. Patients did not receive HSCT as consolidation after CAR-T therapy. Results Thirty-five manufacturing cycles were successful. Median transduction efficacy was 60% (20-80). Median expansion of T cells was x 46 (18-51). CD4:CD8 ratio in the final product was 0.73. The cell products were administered at a dose of 3*106/kg of CAR-T cells in 4 pts, 1*106/kg in 9 pts, 0.5*106/kg in 14 pts, 0.1*106/kg in 8 pts. Two patients received 0.1*106/kg of CAR-T cells produced from haploidentical donors. The cytokine release syndrome (CRS) occurred in 22 (59%) pts and was mostly mild and moderate: grade I - 15 pts, grade II- 4 pts, grade III - 2 pt, grade IV - 1 pt. CAR-T cell related encephalopathy occurred in 15 (40%). Grade I-II neurotoxicity developed in 10 pts, grade III - in 2 pt, grade IV - 1 pt, grade V - 2 pt. In one patient with grade V neurotoxicity concomitant K. pneumonia encephalitis was documented. Severe (grade 3-5) CRS and neurotoxicity were associated exclusively with large leukemia burden (>20% in the bone marrow) at enrollment, p=0,002. Thirty-one patient was evaluable for response at day 28. Four pts had persistent leukemia. In 27 (87%) cases Flow MRD-negative remission was achieved. Disease relapse after initial response was registered in 9 (33%) cases (7 patients had CD19 negative, 2 had CD19 positive relapse). At the moment of reporting, 10 patients have died (3 due to sepsis, 1 due to brain edema, 1 due to brain edema and K. pneumonia encephalitis, 5 due to progression of disease or relapse). Twenty-seven pts are alive, 19 in complete remission with a median follow up of 223 days (41-516 days). Conclusion CliniMACS Prodigy TCT process is a robust CAR-T cell manufacturing platform that enables rapid and flexible provision of CAR-T cells to patients in need. Significant toxicity of CD19 CAR-T cells was associated exclusively with high leukemia burden at enrollment. In the absence of HSCT consolidation relapse rate exceeds 30%. Disclosures Schneider: Lentigen Technology, A Miltenyi Biotec Company: Employment. Preussner:Miltenyi Biotec: Employment. Rauser:Miltenyi Biotec: Employment. Orentas:Lentigen Technology Inc., a Miltenyi Biotec Company: . Dropulic:Lentigen Technology, A Miltenyi Biotec Company: Employment. Maschan:Miltenyi Biotec: Other: lecture fee.
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Gaja, Vijay, Jacqueline Cawthray, Clarence R. Geyer, and Humphrey Fonge. "Production and Semi-Automated Processing of 89Zr Using a Commercially Available TRASIS MiniAiO Module." Molecules 25, no. 11 (June 5, 2020): 2626. http://dx.doi.org/10.3390/molecules25112626.
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The increased interest in 89Zr-labelled immunoPET imaging probes for use in preclinical and clinical studies has led to a rising demand for the isotope. The highly penetrating 511 and 909 keV photons emitted by 89Zr deliver an undesirably high radiation dose, which makes it difficult to produce large amounts manually. Additionally, there is a growing demand for Good Manufacturing Practices (GMP)-grade radionuclides for clinical applications. In this study, we have adopted the commercially available TRASIS mini AllinOne (miniAiO) automated synthesis unit to achieve efficient and reproducible batches of 89Zr. This automated module is used for the target dissolution and separation of 89Zr from the yttrium target material. The 89Zr is eluted with a very small volume of oxalic acid (1.5 mL) directly over the sterile filter into the final vial. Using this sophisticated automated purification method, we obtained satisfactory amount of 89Zr in high radionuclidic and radiochemical purities in excess of 99.99%. The specific activity of three production batches were calculated and was found to be in the range of 1351–2323 MBq/µmol. ICP-MS analysis of final solutions showed impurity levels always below 1 ppm.
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Arrasate, Ane, Igone Bravo, Carlos Lopez-Robles, Ane Arbelaiz-Sarasola, Maddi Ugalde, Martha Lucia Meijueiro, Miren Zuazo, et al. "Establishment and Characterization of a Stable Producer Cell Line Generation Platform for the Manufacturing of Clinical-Grade Lentiviral Vectors." Biomedicines 12, no. 10 (October 4, 2024): 2265. http://dx.doi.org/10.3390/biomedicines12102265.
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Background/Objectives: To date, nearly 300 lentiviral-based gene therapy clinical trials have been conducted, with eight therapies receiving regulatory approval for commercialization. These advances, along with the increased number of advanced-phase clinical trials, have prompted contract development and manufacturing organizations (CDMOs) to develop innovative strategies to address the growing demand for large-scale batches of lentiviral vectors (LVVs). Consequently, manufacturers have focused on optimizing processes under good manufacturing practices (GMPs) to improve cost-efficiency, increase process robustness, and ensure regulatory compliance. Nowadays, the LVV production process mainly relies on the transient transfection of four plasmids encoding for the lentiviral helper genes and the transgene. While this method is efficient at small scales and has also proven to be scalable, the industry is exploring alternative processes due to the high cost of GMP reagents, and the batch-to-batch variability predominantly attributed to the transfection step. Methods: Here, we report the development and implementation of a reliable and clinical-grade envisioned platform based on the generation of stable producer cell lines (SCLs) from an initial well-characterized lentiviral packaging cell line (PCL). Results: This platform enables the production of VSV-G-pseudotyped LVVs through a fully transfection-free manufacturing process. Our data demonstrate that the developed platform will facilitate successful technological transfer to large-scale LVV production for clinical application. Conclusions: With this simple and robust stable cell line generation strategy, we address key concerns associated with the costs and reproducibility of current manufacturing processes.
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Becker, Fabienne, Rigveda Bhave, Soraia Martins, Melanie Hühne, Boris Greber, and Gesine Kogler. "Abstract 12: Cord Blood Banking, Technical and Clinical grade GMP- Development of Advanced Therapy Products as HLA-Homozygous iPSC-derived Cardiomyocytes." Stem Cells Translational Medicine 13, Supplement_1 (August 21, 2024): S14. http://dx.doi.org/10.1093/stcltm/szae062.012.
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Abstract Introduction The Cord Blood Bank (CBB) in Düsseldorf stores 21477 active cryopreserved cord blood units (CBUs) licensed by the Paul-Ehrlich Institute, with 1517 transplants delivered worldwide. We have previously documented a stable storage time of 29 years in liquid nitrogen (Liedtke et al. 2024). Since establishing confirmatory typing and high-resolution HLA sequencing for CBUs and segments in 2016, the CBB progressed into advanced cellular therapies with high-qualified source material and applicable grants (BMBF-161B0760B and HEAL-101056712). Production permission for isolation and short-term expansion of CD34+ cells from licensed CBUs was granted, allowing the GMP reprogramming of 17 HLA-homozygous lines in collaboration with CATALENT (Terheyden-Keighley et al. 2024, accepted in SCTM). Objectives As part of the HEAL project, CD34+ HSC-derived iPSCs were differentiated into cardiomyocytes (CM) using suspension cultures to produce CM-aggregates instead of single cells, as studies in non-human primates indicate better engraftment of aggregates than single cells (Gruh et al. 2024). An optimized cryopreservation process for CM-aggregates is under development applying techniques available to CBU banking. Methods iPSCs were differentiated into CM-aggregates via an improved WNT pathway modulation protocol in suspension culture. In addition to the WNT inhibitor CHIR99021, BMP, Activin A and FGF-2 were added to target accessory pathways and achieve robust scalability across different platforms. Following differentiation, the CM-aggregates were cryopreserved in 10% DMSO with HSA or KO-SR supplementation using a controlled-rate freezer adapted to tissue freezing to ensure uniform freezing processes. Results The improved protocol allowed for straightforward upscaling of CM-aggregate differentiation from 6-well plates to Erlenmeyer flasks of different volumes, leading to a 1-2 fold yield of CM-aggregates with a purity of up to 95 % cardiac troponin T (cTnT) and sarcomeric actinin (sAct). Post-cryopreservation recovery of aggregates was between 60-90% with a viability of ~90%. Although CM culture after thawing remains problematic (<60% CM recovery after 5 days), the aggregates restored their morphology and function. Discussion The CBU banking, processing and storage should meet quality and release criteria, including genetic testing, allowing both allogeneic and related CBU selection to provide the best possible starting material. These advanced therapy products can be utilized e.g. for cardiomyocyte production for potential clinical applications. Overview of a GMP-process development for advanced therapy products from selected HLA-homozygous Cord Blood Units
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Ventura, A., P. Palmerini, A. Dalla Pietà, R. Sommaggio, G. Astori, K. Chieregato, M. Tisi, et al. "P09.13 Optimization of a GMP-grade large-scale expansion protocol for cytokine-induced killer cells using gas-permeable static culture flasks." Journal for ImmunoTherapy of Cancer 8, Suppl 2 (October 2020): A58.2—A59. http://dx.doi.org/10.1136/jitc-2020-itoc7.113.
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BackgroundCytokine-Induced Killer (CIK) cells are ex vivo expanded T cells with NK cell phenotype. They express both CD3 and CD56 antigens, and exert a potent antitumor activity against a variety of tumors. Several clinical trials demonstrated the safety and the feasibility of CIK cell therapy, with very low side effects and minimal graft-versus-host toxicity. In this study, we developed a GMP-compliant protocol for robust large-scale expansion of CIK cells using G-Rex® gas-permeable static culture flasks.Materials and MethodsCIK cells were obtained by stimulating healthy donor PBMCs with GMP-grade IFN-γ, IL-2 and CD3 mAbs, and were cultured in G-Rex6® or G-Rex®6M well plates. CIK cells in G-Rex6® were split only once at day 7 to reduce cell density, whereas the number of CIK cells culterd in G-Rex®6M was not adjusted. In both culture conditions, fresh IL-2 was provided every 3–4 days. We compared these two culture protocols with the culture in standard flasks. Phenotype was analyzed by flow cytometry and cytotoxicity was assessed against several tumor cell lines by calcein-release assay.ResultsCIK cells cultured in G-Rex6® well plates showed an outstanding cell expansion compared to G-Rex®6M well plates or standard culture flasks, with a 400-fold expansion and a mean of 109 total cells obtained per single well in 14 days, starting from just 2.5 × 106 cells per well. Moreover, the cultures in G-Rex6® were characterized by an higher percentage of CD3+CD56+ cells, as compared to G-Rex®6M or standard culture flasks. Cells cultured in all devices had a comparable expression of NKG2D, NKp30, NKp44, 2B4 receptors. Importantly, CIK cells expanded in G-Rex®6 were as cytotoxic as cells expanded in standard culture flasks. Conversely, CIK cells cultured in G-Rex®6M showed a remarkable reduction of cytotoxicity against tumor cell targets, thus suggesting that cell density during expansion could affect CIK cell activity.ConclusionsWe propose a GMP-compliant protocol for robust large-scale production of CIK cells. G-Rex® system allows to obtain large amounts of CIK cells highly enriched in the CD3+CD56+ subset and endowed with high cytotoxic activity; this can be accomplished with just a single cell culture split at day 7, which dramatically reduces the culture manipulation as compared to the standard culture flasks. Notably, this strategy can be further and easily scalable to produce CIK cells for clinical immunotherapy applications.Disclosure InformationA. Ventura: None. P. Palmerini: None. A. Dalla Pietà: None. R. Sommaggio: None. G. Astori: None. K. Chieregato: None. M. Tisi: None. C. Visco: None. O. Perbellini: None. M. Ruggeri: None. E. Cappuzzello: None. A. Rosato: None.
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Fadeyev, F. A., D. V. Lugovets, M. V. Ulitko, S. L. Leontyev, and S. V. Sazonov. "The dependence of proliferation rate of human dermal fibroblasts on growth medium composition and fetal bovine serum concentration." Genes & Cells 11, no. 4 (December 15, 2016): 75–79. http://dx.doi.org/10.23868/gc120588.
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Application of dermal fibroblasts for therapy requires the use of large volume of cell material. The automatized cultivation provides stable conditions for GMP-grade cell production. To define the effect of serum concentration on proliferation rate, fibroblasts were cultured in growth media with different serum percentage. The proliferation rate was evaluated by the use of average period of cell population doubling. For choosing the optimal culture medium, different types of media together with their mixtures, produced by Gibco and PanEco, were explored. It was shown that exponential decrease of the period of cell population doubling occurs when the serum concentration rises from zero to 1 2%, and further increment of serum percentage is followed only by minor reduction of this value. The highest proliferation rate of dermal fibroblasts is obtained when they are cultivated in aMEM medium or in the mixtures of aMEM with F-12, Advanced DMEM with F-12 and Advanced DMEM with RPMI-1640 media (Gibco, USA).
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Hörmann, Anton Amadeus, Gregor Schweighofer-Zwink, Gundula Rendl, Kristina Türk, Samuel Nadeje, Kristina Haas, Theresa Jung, et al. "[68Ga]Ga-FAP-2286—Synthesis, Quality Control and Comparison with [18F]FDG PET/CT in a Patient with Suspected Cholangiocellular Carcinoma." Pharmaceuticals 17, no. 9 (August 29, 2024): 1141. http://dx.doi.org/10.3390/ph17091141.
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[68Ga]Ga-FAP-2286 is a new peptide-based radiopharmaceutical for positron-emission tomography (PET) that targets fibroblast activation protein (FAP). This article describes in detail the automated synthesis of [68Ga]Ga-FAP-2286 using a commercially available synthesis tool that includes quality control for routine clinical applications. The synthesis was performed using a Scintomics GRP-3V module and a GMP grade 68Ge/68Ga generator. A minor alteration for transferring the eluate to the module was established, eliminating the need for new method programming. Five batches of [68Ga]Ga-FAP-2286 were tested to validate the synthesis. A stability analysis was conducted up to 3 h after production to determine the shelf-life of the finished product. The automated synthesis on the Scintomics GRP-3V synthesis module was found to be compliant with all quality control requirements. The shelf-life of the product was set to 2 h post-production based on the stability study. A patient suffering from cholangiocellular carcinoma that could not be clearly detected by conventional imaging, including a [18F]FDG-PET/CT, highlights the potential use of [68Ga]Ga-FAP-PET/CT.
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Xiang, Shuanglin, Wenyong Gao, Haining Peng, Aibing Liu, Qiang Ao, Manjun Yang, Yanqiu Yu, Ying Liu, Raoxing Rong, and Raoxing Rong. "Standards of clinical-grade mesenchymal stromal cell preparation and quality control (2020 China Version)." Journal of Neurorestoratology 8, no. 4 (2020): 197–216. http://dx.doi.org/10.26599/jnr.2020.9040021.
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Mesenchymal stromal cells (MSCs) including mesenchymal stem cells to potentially differentiate into different tissue lineages widely exist in various tissues. In recent years, the clinical research and application of MSCs have become more extensive, but no standardized guidelines for the preparation and quality control of clinical-grade MSCs currently exist. To standardize the preparation and quality control of MSCs using the human umbilical cord, placenta, bone marrow, and adipose tissue as sample sources for the Chinese Association of Neurorestoratology (CANR; Preparatory) and the China Committee of International Association of Neurorestoratology (IANR-China Committee) member units, this standard is formulated following the T11/CSSCR 001-2017 General Requirements for Stem Cells, Good Manufacturing Practice Pharmaceutical Products (2010 Edition), Pharmacopoeia of the People's Republic of China (2015 Edition), Guiding Principles for Quality Control of Stem Cell Preparations and Preclinical Research (Trial), Code for Cell Banking Facility Quality Management, Sterile Drug Appendix to Pharmaceutical Production Quality Management Regulations, GMP Appendix — Cell Therapy Products (Draft for Comment), The International Society for Cellular Therapy position statement (2006), and Clinical Cell Therapy Guidelines for Neurorestoration (IANR/CANR 2017). Moreover, this standard includes donor evaluation, sample collection, cell preparation, cell inspection, packaging, labeling, transportation and storage, and quality control. It represents the minimum requirements for clinical-grade mesenchymal stromal cell culture and quality control. Moreover, it will be further optimized following the progress of preclinical and clinical research.
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Moreau, Thomas, Maria Colzani, Meera Arumugam, Amanda Evans, Marloes Renee Tijssen, Matthew Trotter, Willem H. Ouwehand, Roger Pedersen, and Cedric Ghevaert. "In Vitro Production Of Megakaryocytes and Platelets From Human Induced Pluripotent Cells By GMP Compatible Methods." Blood 122, no. 21 (November 15, 2013): 2401. http://dx.doi.org/10.1182/blood.v122.21.2401.2401.
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Abstract Human pluripotent stem cells and in particular induced pluripotent stem cells (iPSC) derived from adult tissue have recently opened novel and promising avenues for cell therapy. In vitro production of high demand transfusion products such as platelets from HLA- and HPA-typed iPSC lines is an attractive target but to achieve this potential there is a clear need for reliable and efficient GMP compliant differentiation protocols and dedicated cell culture devices compatible with large scale cell production. We first developed a novel protocol for production of megakaryocytes (MKs) from human iPSCs based on the ectopic expression of specific transcription factors (TFs), so called “forward programming”. Through a transcriptome and protein interactome guided process, we initially shortlisted 46 TF candidates and eventually tested fourteen of the top ranked factors for their forward programming potential. Using lentiviral vectors we identified a minimal combination of three TFs which generated the highest amount of MKs. This forward programming protocol was able to robustly induce MK differentiation from various embryonic and iPSC lines. Further developments have lead to a fully standardise differentiation system using forced aggregation of defined amounts of single iPSCs into embryoid bodies and chemically defined media throughout the culture. We reliably achieve a 99% pure MK culture with a mature phenotype (i.e. CD41a+/CD61+/CD42a+/CD42b+/GP6+, polyploid and forming proplatelets and expressing key regulatory genes (e.g. NFE2, MEIS1, PBX1, RUNX1, ZFPM1) and a cell yield in excess of 50 times the initial iPSC input. These results surpass existing protocols offering wider perspectives in using human iPSC derived MKs for biological studies as well as clinical applications. This field of research has also been hampered by the lack of efficient, GMP-compatible protocols to derive platelets from MKs. We set out to address this issue by creating a 3-dimensional biomimetic/biocompatible scaffold to be incorporated a perfusion bioreactor to mimic the chemico-mechanical signals to enhance proplatelet formation and platelet release from MKs and allow the harvest of functional platelets in numbers compatible with those required for human use. The importance of the niche environment (including cell-to-cell contact and extracellular matrix proteins) for both MK maturation and platelet release has been demonstrated in different studies. Based on protein expression data from endothelial cells and mesenchymal cell lines that support platelet formation in co-cultures, we have generated a library of recombinant transmembrane proteins (TMPs). These TMPs contain the extracellular domain of the candidate transmembrane proteins at the N-terminal while at the C-terminus they contain a sequence which allows purification and immobilization on either culture plates or 3D scaffolds. We have used a combinatorial approach to perform a high throughput screening of the TMP library identify which TMPs promote proplatelet formation. Briefly, TMPs were immobilized onto 96-well plates onto which mature culture-derived MKs were seeded and proplatelet formation recorded by microscopy and digital imaging analysis. The best candidate combinations were identified through a mathematical model adapted from drug screening experiments. Finally we have produced porous matrices using GMP-grade collagen and fibrinogen via a freeze-drying process whereby ice crystals introduced into a protein-acid slurry are sublimated off to produce a fully interconnected porous structure. The structure can be tailored in terms of pore sizes and morphologies by varying the temperature profile. The structural integrity over prolonged periods in tissue culture can be enhanced by chemical crosslinking, which also removes the active moiety of collagen. We show that both collagen and fibrinogen scaffolds support MK culture from cord-blood derived CD34+ stem cells with no toxic effects and similar differentiation properties as with standard 2D cultures. We also show that the scaffold support platelet formation from seeded mature MKs. These inert scaffolds can be further functionalized by affixing the candidate TMPs identified above via a UV-light driven process onto the inert base scaffold to give a direct contact signal to the MKs to produce platelets. Disclosures: No relevant conflicts of interest to declare.
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Montemurro,, Tiziana, Cristiana Lavazza, Gabriella Andriolo, Mariele Viganò, Barbara Baluce, Paolo Rebulla, Rosaria Giordano, and Lorenza Lazzari. "Off-the-Shelf Cord Blood Mesenchymal Stem Cell Bank." Blood 118, no. 21 (November 18, 2011): 4403. http://dx.doi.org/10.1182/blood.v118.21.4403.4403.
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Abstract Abstract 4403 Our hospital-based GMP facility, named Cell Factory, built in Milano (Italy) and the first one authorized by the National Drug Agency (AIFA) in 2007, has pioneered innovative manufacturing technologies in order to supply therapeutic clinical development programs. By our group and by others, cord blood (CB) mesenchymal stem cells (MSC) have been already tested both in vitro and in extensive pre-clinical disease models, demonstrating that CBMSC are safe and effective. Therefore, CBMSC are the best candidate product for several clinical trials including neurologic and renal repair (1–3). In this context, our group developed the first off-the-shelf (OTS) bank where CBMSC are expanded and banked in GMP conditions, and now they are available as “a drug”, to be distributed for patient doses, much like an off-the-shelf pharmaceutical product. From informed consent CB donations, CBMSC were grown and expanded in CellSTACK kit that includes devices for a closed-system cell culture (Macopharma) with EMEA-grade Australian FBS and they were manufactured through a multi-step controlled and well-validated process. Cell Factory established this innovative culture approach through sufficient passages to produce hundreds of thousands of potential doses from an individual donor. After culture, our quality control approach has been designed to ensure that controls are implemented and completed satisfactorily during manufacturing operations, and that developed procedures and specifications are appropriate and followed, including those performed by contract testing laboratories. In fact, at each step and at the end of the production process before cryopreservation, CBMSC were analyzed and qualified according to pre-established criteria (cell count, viability, extensive immunophenotype, CFU-F, karyotype, CGHArray, telomerase activity, sterility, mycoplasma and endotoxin assays) to ensure that a consistent, well characterized product candidate is produced. The same CBMSC batches can be provided for pre-clinical testing and for further validations to prepare the investigational medicinal product dossier (IMPD) for each clinical trial approvals. Now these GMP batches of CBMSC are cryopreserved and available in our OTS CBMSC bank in different cell doses to comply any kind of medical needs. Products are provided under specific contract conditions developed with the clinical centers. (Contact: cellfactorypoliclinico@gmail.com) Disclosures: No relevant conflicts of interest to declare.
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Spaccapaniccia, Elisa, Tiziano Cazzorla, Daniela Rossetti, Lucio De Simone, Maria Irene Antonangeli, Andrea Antonosante, Francesca Galli, Franca Cattani, Mariano Maffei, and Franck Martin. "Efficient Production of Recombinant Human Brain-Derived Neurotrophic Factor in Escherichia coli Through the Engineering of Its Pro-Region." International Journal of Molecular Sciences 25, no. 24 (December 14, 2024): 13425. https://doi.org/10.3390/ijms252413425.
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Thus far, no manufacturing process able to support industrialization has been reported for the recombinant human brain-derived neurotrophic factor (rhBDNF). Here, we described the setup of a new protocol for its production in Escherichia coli (E. coli) and its purification to homogeneity. A synthetic gene, codifying for the neurotrophin precursor, was inserted into an E. coli expression vector and transformed into BL21 (DE3) strain. The recombinant protein accumulates, at high yields, into inclusion bodies. With the developed strategy, more than 50% of the precursor can be refolded. The protein is successively digested by trypsin and the rhBDNF mature form is finally purified by two additional chromatographic steps If the wild-type precursor can be efficiently obtained by the proposed methodology, its pro-peptide remotion, through enzymatic digestion, is however problematic. To circumvent this difficulty, the precursor hinge region, containing the natural furin recognition site, was engineered to be more specifically cleaved by trypsin. Notwithstanding the substitution of three residues in the pro-region carboxyterminal, the precursor correctly refolds and is efficiently cleaved to generate a biologically active mature rhBDNF. This efficient high-yield process fills the current need of a scalable protocol to produce GMP-grade material and unlocks the rhBDNF employment in future clinical investigations.
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Gaipa, Giuseppe, Chiara Francesca Magnani, Daniela Belotti, Giada Matera, Sarah Tettamanti, Benedetta Cabiati, Stefania Cesana, et al. "Clinical-Grade Transduction of Allogeneic Cytokine Induced Killer (CIK) Cells with CD19 Chimeric Antigen Receptor (CAR) Using Sleeping Beauty (SB) Transposon: Successful GMP-Compliant Manufacturing for Clinical Applications." Blood 132, Supplement 1 (November 29, 2018): 196. http://dx.doi.org/10.1182/blood-2018-196.
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Abstract Background: Acute lymphoblastic leukemia (ALL) is a malignant disorder with a long-term remission of less than 50% of adult patients and of nearly 80% of children. Relapsed and refractory (r/r) adult and childhood B-ALL patients, have significant unmet medical needs. Adoptive transfer of patient-derived T cells engineered to express a chimeric antigen receptor (CAR) by viral vectors has achieved complete remission and durable response in highly refractory populations (June CH et al. Science 2018). In addition, unmodified Cytokine Induced Killer (CIK) cells (CD3+, CD56+ T cells) have clearly demonstrated a high profile of safety in ALL patients (Introna M et al. Biol Blood Marrow Transplant. 2017). Here, we demonstrate the feasibility and reproducibility of a GMP-compliant clinical-grade culture and gene-modification protocol of allogeneic CIK cells using the non-viral Sleeping Beauty (SB) transposon system (Singh H et al, Plos One 2013) to obtain CD19CAR expressing CIK cells (Magnani CF et al, Oncotarget 2016, Magnani CF et al, Hum Gene Ther. 2018, Biondi A et al. J Autoimmun. 2017) starting from a limited amount of an easily available material such as peripheral blood (PB). Methods: Fifty mL of PB were centrifuged on Ficoll gradient to obtain mononuclear cells (PBMCs). PBMCs were then simultaneously electro-transferred with the SB GMP-grade DNA transfer CD19.CAR/pTMNDU3 plasmid (human 3rd generation anti-CD19CD28OX40z CAR under the pTMNDU3 promoter), and transposase pCMV-SB11 plasmid (kindly provided by L. Cooper, MDACC, Houston, TX, USA). CIK populations (Introna M et al, Haematologica 2007) were then generated according to the method enclosed in the filed patent EP20140192371 (Magnani CF et al, Oncotarget 2016). The manufacturing process and the quality control tests were performed in a good manufacturing practices (GMP) academic cell factory authorized by Agenzia Italiana del Farmaco (AIFA) in the context of an ongoing phase I clinical trial (NCT03389035) for children and adults with relapsed/refractory B-cell precursor ALL post hematopoietic stem cell transplantation (HSCT). Results: We manufactured nine batches by seeding a mean of 102.52x106 allogeneicPBMCs derived from 50 ml of peripheral blood (range 46.1 - 193.17x106). After 21-22 days of culture the mean harvesting was 5.0x109 nucleated cells (range 1.15 - 16.00x109) with a mean viability of 97.56% (min. 95.24% - max 99.43%). These cells were mostly CD3+ lymphocytes (mean 98.54%, min. 94.85% - max 99.68%) which had a median fold increase of 87.3. Expanded CD3+ cells expressed CD56+ and surface CAR at variable levels among the batches (mean 44.79% and 43.78%, respectively) and had a median vector copy number (VCN) of 2.56 VCN/cells, according to pre-clinical data (Magnani CF et al, Hum Gene Ther. 2018). In all the nine batches, CARCIK-CD19 cells demonstrated potent and specific in vitro cytotoxicity towards the CD19+ REH target cell line (mean 82.96%, min. 61.89% - max 97.72%). Cell products appear to be highly polyclonal and no signs of genotoxicity by transposon insertions could be observed by integration site (IS) analysis performed by Sonication Linker Mediated (SLiM)-PCR and Illumina MiSeq sequencing. The GMP batches were released after about 10 days after the end of production. Quality control release specifications and results are reported in Table 1. Conclusions: Overall, these results demonstrate that clinical-grade SB transduction of allogeneic CIK cells with CD19 CAR is feasible and allows rapid and efficient expansion of highly potent CARCIK-CD19 cells starting from easily available small amounts of PB, with important implications for non-viral technology. In summary our data represent a solid ground for the future development of further SB-based platforms. A clinical trial investigating allogeneic CARCIK-CD19 in r/r pediatric and adult ALL post HSCT is currently ongoing (NCT03389035). Disclosures Gritti: Autolus: Consultancy. Rambaldi:Celgene: Consultancy; Omeros: Consultancy; Novartis: Consultancy; Italfarmaco: Consultancy; Pfizer: Consultancy; Amgen Inc.: Consultancy; Roche: Consultancy.
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Wright, Adrienne, Orman L. Snyder, Lane K. Christenson, Hong He, and Mark L. Weiss. "Effect of Pre-Processing Storage Condition of Cell Culture-Conditioned Medium on Extracellular Vesicles Derived from Human Umbilical Cord-Derived Mesenchymal Stromal Cells." International Journal of Molecular Sciences 23, no. 14 (July 13, 2022): 7716. http://dx.doi.org/10.3390/ijms23147716.
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EVs can be isolated from a conditioned medium derived from mesenchymal stromal cells (MSCs), yet the effect of the pre-processing storage condition of the cell culture-conditioned medium prior to EV isolation is not well-understood. Since MSCs are already in clinical trials, the GMP-grade of the medium which is derived from their manufacturing might have the utility for preclinical testing, and perhaps, for clinical translation, so the impact of pre-processing storage condition on EV isolation is a barrier for utilization of this MSC manufacturing by-product. To address this problem, the effects of the pre-processing storage conditions on EV isolation, characterization, and function were assessed using a conditioned medium (CM) derived from human umbilical cord-derived MSCs (HUC-MSCs). Hypothesis: The comparison of three different pre-processing storage conditions of CM immediately processed for EV isolation would reveal differences in EVs, and thus, suggest an optimal pre-processing storage condition. The results showed that EVs derived from a CM stored at room temperature, 4 °C, −20 °C, and −80 °C for at least one week were not grossly different from EVs isolated from the CM immediately after collection. EVs derived from an in pre-processing −80 °C storage condition had a significantly reduced polydispersity index, and significantly enhanced dot blot staining, but their zeta potential, hydrodynamic size, morphology and size in transmission electron microscopy were not significantly different from EVs derived from the CM immediately processed for isolation. There was no impact of pre-processing storage condition on the proliferation of sarcoma cell lines exposed to EVs. These data suggest that the CM produced during GMP-manufacturing of MSCs for clinical applications might be stored at −80 °C prior to EV isolation, and this may enable production scale-up, and thus, and enable preclinical and clinical testing, and EV lot qualification.
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Seetharaman, Abirami, Vasanth Christopher, Hemavathi Dhandapani, Hascitha Jayakumar, Manikandan Dhanushkodi, Narmadha Bhaskaran, Swaminathan Rajaraman, et al. "Optimization and Validation of a Harmonized Protocol for Generating Therapeutic-Grade Dendritic Cells in a Randomized Phase II Clinical Trial, Using Two Varied Antigenic Sources." Vaccines 12, no. 2 (January 23, 2024): 112. http://dx.doi.org/10.3390/vaccines12020112.
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Autologous dendritic cell (DC)-based immunotherapy is a cell-based advanced therapy medicinal product (ATMP) that was first introduced more than three decades ago. In the current study, our objective was to establish a harmonized protocol using two varied antigenic sources and a good manufacturing practice (GMP)-compliant, manual method for generating clinical-grade DCs at a limited-resource academic setting. After obtaining ethical committee-approved informed consent, the recruited patients underwent leukapheresis, and single-batch DC production was carried out. Using responder-independent flow cytometric assays as quality control (QC) criteria, we propose a differentiation and maturation index (DI and MI, respectively), calculated with the QC cut-off and actual scores of each batch for comparison. Changes during cryopreservation and personnel variation were assessed periodically for up to two to three years. Using our harmonized batch production protocol, the average DI was 1.39 and MI was 1.25. Allogenic responder proliferation was observed in all patients, while IFN-gamma secretion, evaluated using flow cytometry, was detected in 10/36 patients and significantly correlated with CD8+ T cell proliferation (p value-0.0002). Tracking the viability and phenotype of cryopreserved MDCs showed a >90% viability for up to three years, while a mature DC phenotype was retained for up to one year. Our results confirm that the manual/semi-automated protocol was simple, consistent, and cost-effective, without the requirement for expensive equipment and without compromising on the quality of the final product.
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Belotti, Daniela, Giuseppe Gaipa, Beatrice Bassetti, Benedetta Cabiati, Gabriella Spaltro, Ettore Biagi, Matteo Parma, et al. "Full GMP-Compliant Validation of Bone Marrow-Derived Human CD133+Cells as Advanced Therapy Medicinal Product for Refractory Ischemic Cardiomyopathy." BioMed Research International 2015 (2015): 1–10. http://dx.doi.org/10.1155/2015/473159.
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According to the European Medicine Agency (EMA) regulatory frameworks, Advanced Therapy Medicinal Products (ATMP) represent a new category of drugs in which the active ingredient consists of cells, genes, or tissues. ATMP-CD133 has been widely investigated in controlled clinical trials for cardiovascular diseases, making CD133+cells one of the most well characterized cell-derived drugs in this field. To ensure high quality and safety standards for clinical use, the manufacturing process must be accomplished in certified facilities following standard operative procedures (SOPs). In the present work, we report the fully compliant GMP-grade production of ATMP-CD133 which aims to address the treatment of chronic refractory ischemic heart failure. Starting from bone marrow (BM), ATMP-CD133 manufacturing output yielded a median of 6.66 × 106of CD133+cells (range 2.85 × 106–30.84 × 106), with a viability ranged between 96,03% and 99,97% (median 99,87%) and a median purity of CD133+cells of 90,60% (range 81,40%–96,20%). Based on these results we defined our final release criteria for ATMP-CD133: purity ≥ 70%, viability ≥ 80%, cellularity between 1 and 12 × 106cells, sterile, and endotoxin-free. The abovementioned criteria are currently applied in our Phase I clinical trial (RECARDIO Trial).
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Krampera, Mauro, Cedric Mènard, Luciano Pacelli, Giulio Bassi, Joelle Dulong, Isabelle Bezier, Francesco Bifari, et al. "Quality Controls of Immune Regulatory Properties of Ex-Vivo, GMP-Grade Expanded Mesenchymal Stromal Cells for Clinical Use (European multicenter study CASCADE),." Blood 118, no. 21 (November 18, 2011): 4049. http://dx.doi.org/10.1182/blood.v118.21.4049.4049.
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Abstract Abstract 4049 Aim of the European Consortium CASCADE is to standardize GMP-grade production and clinical use of Mesenchymal Stromal Cells (MSC) to treat skin and corneal wounds. MSC possess immunogenicity and immunomodulatory properties that must be carefully addressed before clinical use. CASCADE Immunological Unit is aimed to set up and validate a wide panel of functional assays to fully characterize in a standardized and reproducible manner the immunomodulatory properties of MSC obtained inside CASCADE Units from bone marrow, adipose tissue, cord blood, and amniotic membrane (BM, AT, CB, AM) through different GMP-grade expansion protocols including platelet lysate- and fetal calf serum-based culture conditions. Immune cells were isolated using indirect immunomagnetic depletion; samples with less than 96% of purity were discarded. For the experiments, MSC were expanded in the same medium used for production and harvested at 70% confluence. Primed MSC were obtained by 48h-treatment with 10 ng/ml of rh-INFγ and 15 ng/ml of rh-TNFα. Cocultures were set up by plating primed or unprimed MSC in 96 or 48 flat bottomed – well plates; CFSE-stained T, B, NK cells were seeded at different effector cell:MSC ratios. Cells were harvested after 4 or 6 days of coculture for proliferation evaluation by FACS analysis. T cells were stimulated with mitogenic αCD3 plus αCD28 antibodies at 0.5 μg/ml each; B cells were activated with CD40L at 50 ng/ml, its enhancer at 5 g/ml, IL-2 20 UI/ml, CpG 2006 2.5 μg/mL, and F(ab')2 anti-IgM/IgA/IgG 2 μg/mL; NK cells were activated with 100 U/ml rh-IL2. To identify the molecular mechanisms involved in immunomodulatory properties of MSC, coculture of immune effector cells and MSC were performed in the presence of specific inhibitors, after identifying their non-toxic and effective concentrations: 1 mM for L-1MT (IDO inhibitor), 2 μM for snPP (HO-1 inhibitor), 5 μM for NS-398 (COX2 inhibitor), 1 mM for L-NMMA (iNOS inhibitor) and 10 μg/ml for anti-IFNγ neutralizing antibody. We also studied the capacity of resting and primed MSC to sustain the survival of unstimulated T, B, and NK cells through the evaluation of the percentage of caspase-3negCD45pos viable immune cells after 4 to 6 days in culture with or without MSC. For MSC immunogenicity assay, the proliferation of allogeneic T was evaluated at day 5 of culture by incorporation of 3H-Thymidine; in addition, NK cells were activated for 2 days with 100 U/ml of rh-IL2 whereas resting or primed MSC were loaded with non radioactive fluorophore (BaTDA) or with Cr51 and used as target cells. Inflammatory milieu significantly upregulated MHC class I and II, CD54, CD106, CD40, CD274, CD112, CD155 expression, and downregulated NKG2D ligands (ULBP 1–3, MICA/B) and mesenchymal markers (CD73, CD90, CD105). AT-derived MSC expressed less MHC class II, CD200 and CD106 molecules than BM-MSC. MSC coculture inhibited T and NK cell proliferation without inducing apoptosis, and this effect was greater in presence of primed MSC. On the contrary, only primed MSC were capable of suppressing B cell proliferation. In addition, MSC inhibited apoptosis of resting T, B, and NK cells, while inflammatory priming increased their pro-survival activity. T cell/MSC coculture showed that activation of IDO and HO-1 was the main mechanism involved in MSC immune modulation, as the addition of specific inhibitors (L-1-MT and snPP) significantly reverted the phenomenon. MSC never promoted allogeneic T cell proliferation; by contrast, IL-2-activated NK cells could efficiently recognize and kill allogenic unprimed MSC. However, MSC became insensitive to NK cells once primed with inflammatory cytokines. Some differences were observed depending on the origin and culture conditions of clinical-grade MSC. All the experimental protocols to assess MSC inhibitory effects on immune effector cells have been standardized and will be applied for the release of GMP-grade MSC produced inside the CASCADE Consortium. Disclosures: No relevant conflicts of interest to declare.
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Liu, Enli, Yijiu Tong, Gianpietro Dotti, Barbara Savoldo, Muharrem Muftuoglu, Kayo Kondo, Malini Mukherjee, et al. "Cord Blood Derived Natural Killer Cells Engineered with a Chimeric Antigen Receptor Targeting CD19 and Expressing IL-15 Have Long Term Persistence and Exert Potent Anti-Leukemia Activity." Blood 126, no. 23 (December 3, 2015): 3091. http://dx.doi.org/10.1182/blood.v126.23.3091.3091.
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Abstract Natural killer (NK) cells are a major component of the innate immune system, possessing the ability to lyse their targets without the need for prior sensitization or specificity for antigen. Besides their classical role in providing potent antitumor and antiviral immunity, NK cells can reduce the risk of graft-versus-host disease (GVHD) by targeting host antigen-presenting cells, as well as activated alloreactive donor T cells, indicating that NK-mediated graft-versus-leukemia (GVL) responses may occur in the absence of GVHD. Although most groups have relied on autologous or adult peripheral blood donor-derived NK cells, we have studied umbilical CB as a potential source of NK cells because of their availability as an "off-the-shelf" frozen product and their potent preclinical activity against leukemia cells. To overcome the obstacle of limited numbers of NK cells in a single CB unit, we have established GMP-compliant conditions for the ex vivo expansion of clinically relevant doses of such cells. By using GMP grade K562-based artificial antigen-presenting cells (aAPCs), which express membrane-bound IL-21 (clone 9.mbIL21), to numerically expand highly functional and mature CB-derived NK cells. To further enhance the GVL effect independent of KIR-ligand mismatch, we have genetically modified human CB-derived NK cells with a retroviral vector, CAR19-CD28-zeta-2A-IL15 (CAR19/IL15), which incorporates the genes for CAR-CD19, IL-15 to enhance proliferation and survival, and the inducible caspase-9 molecule. CB-NK cells could be stably transduced with CAR19/IL15, proliferated efficiently in vitro and maintained superior effector function against CD19-expressing leukemia cell lines and primary CLL cells. Moreover, the effector functions of both NK-CAR and nontransduced NK cells against K562 were comparable, indicating that the genetic modification of CB-NK cells does not alter their intrinsic cytotoxicity against NK-sensitive targets. Because of concerns over autonomous, uncontrolled NK cell growth due to autocrine production of IL15, we also incorporated into our construct a suicide gene based on the inducible caspase-9 (IC9) gene. The addition of as little as 10 nM of the small molecule dimerizer CID AP20187 to cultures of iC9/CAR19/IL15+ NK cells induced apoptosis/necrosis of >60% of transgenic cells within 4 hours as assessed by annexin-V-7AAD staining. The infusion of CAR.CD19.IL15-transduced CB-NK cells into a NOD-SCID-gamma null model of lymphoblastic lymphoma (Raji model) resulted in impressive anti-tumor responses (Fig. 1). Moreover, adoptively infused CAR-transduced CB NK persisted for up to 70 days post-infusion (Fig. 2), supporting our hypothesis that IL-15 enhances the proliferation and survival of the engineered CB-NK cells. Based on these promising data, we now propose to manufacture a GMP-grade CAR19-CD28-zeta-2A-IL15 vector for a phase 1 dose escalation trial in patients with high risk B-cell leukemia. Disclosures Wierda: Celgene Corp.: Consultancy; Glaxo-Smith-Kline Inc.: Research Funding. Rezvani:Pharmacyclics: Research Funding.
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Schlegel, Patrick, Chihab Klose, Christina Kyzirakos, Ursula J. E. Seidel, Kai Witte, Annika Horrer, Marie Matela, Matthias Pfeiffer, Rupert Handgretinger, and Peter Lang. "Transfer of Ex Vivo Expanded NK and γδT Cells from Untouched Posttransplant PBMCs to Clear Minimal Residual Disease in Acute Myeloid Leukemia." Blood 124, no. 21 (December 6, 2014): 5294. http://dx.doi.org/10.1182/blood.v124.21.5294.5294.
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Abstract GMP-grade NK cell expansion for clinical purpose has been demonstrated feasible and safe. Here we share our pilot data on posttransplant immunotherapy with ex vivo expanded NK cells to treat minimal residual disease in a pediatric patient with posttransplant relapsed myeloid leukemia. Our patient, a 13 year old boy who underwent 2nd allogeneic stem cell transplantation (haploidentical stem cell transplantation from his mother) due to posttransplant relapsed acute myeloid leukemia. After the 2nd haploidentical stem cell transplantation (SCT) minimal residual disease (MRD) was detected by multiparameter flow cytometry and by two molecular markers CALM-AF10 fusion transcript and a NRAS-mutation. For posttransplant compassionate use immunotherapy by NK cell transfer, NK cells were expanded from untouched isolated PBMCs of the patient post 2nd haploidentical SCT. GMP-grade expansion of the NK cells was done under static conditions in our GMP-facility. Isolated PBMCs were pooled with 100Gy irradiated K562mb15 4-1BBL feeder cells (kindly provided by Dario Campana) in a proportion of 1:20 (NK to K562mb15 4-1BBL). PBMCs and K562mb15 4-1BBL were seeded in conventional cell culture flasks (175cm2) at a density of 1.1E6 cells/ml. Cell culture media contained RPMI1640 supplemented with 10% AB-human serum, 1% L-glutamine and 100IU Proleukine® IL2/ml. Cell culture was monitored daily for cell number, white blood cell differentiation, pH of the cell culture, glucose metabolism, lactate production and microbial sterility testing at the beginning and the end of the expansion period. The cell product was harvested on day 15-17. Fresh isolated PBMCs and the expanded NK cell product were characterized by flow cytometry. NK cells were expanded >1000 fold (3.1 and 3.4 log-fold) in 14-17 days. The product contained a total number of 9.8E9 and 19.9E9 cells, which was 328 and 665E6/kgBW. The expansion protocol supports NK and γδ T cell expansion whereas the number of αβ T cells stays stable. Cytotoxicity assay against various targets revealed excellent cellular cytotoxicity and antibody dependent cellular cytotoxicity. To prevent relapse in our patient with posttransplant MRD positivity, NK cells from the patient post 2nd haploidentical SCT were expanded for cellular immunotherapy. 2 weeks post 1st NK cell transfer (day +170) the patient achieved complete MRD response in the bone marrow. Unfortunately the patient showed detectable MRD one month later. Therefore another NK cell expansion and transfer was done. 2 weeks post 2nd NK cell transfer (day +232) the patient again achieved complete MRD response in the bone marrow and is in complete molecular remission ever since (day +340). The NK cell products were tolerated well. Transient coughing and temporary increase of temperature were registered. Both, in vitro and in vivo effect of the NK cell product were documented. Clinical use of expanded and activated NK cells and γδ T cells can induce molecular remission in posttransplant MRD positive acute myeloid leukemia. Disclosures No relevant conflicts of interest to declare.
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Ghorashian, Sara, Anne Marijn Kramer, Sarah Jayne Albon, Catherine Irving, Lucas Chan, Fernanda Castro, Bilyana Popova, et al. "A Novel Second Generation CD19 CAR for Therapy of High Risk/Relapsed Paediatric CD19+ Acute Lymphoblastic Leukaemia and Other Haematological Malignancies: Preliminary Results from the Carpall Study." Blood 128, no. 22 (December 2, 2016): 4026. http://dx.doi.org/10.1182/blood.v128.22.4026.4026.
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Abstract Introduction: Recent clinical trials with T cells engineered to express 2nd generation CD19 chimeric antigen receptors (CARs) unprecedented anti-leukemic responses. We have developed a novel CD19CAR with a new scFv in the 41BBz format (CAT-41BBz CAR) which confers enhanced cytotoxicity and cytokine secretion in response to stimulation with CD19+ targets in vitro as well as equivalent in vivo anti-tumour efficacy to the FMC63 41BBZ CAR in use in clinical studies. We have designed, optimized and validated GMP-grade CAR T cell production using this novel CAR. Based on these data, we have recently initiated a Phase I clinical study (CARPALL) of this novel CAR in pediatric patients with relapsed ALL and other CD19+ hematological malignancies to determine the safety profile and durability of responses to CD19CART therapy. This will be critical in determining whether CD19CAR T cells are best used as a stand-alone therapy or as a bridge to stem cell transplant (SCT). Methods: We initially optimized our GMP production methodology in terms of activation method, cytokine milieu and expansion conditions on healthy donor peripheral blood mononuclear cells (PBMCs) to give optimal transduction efficiency and preserve early memory subsets within the CAR T cell product. We have subsequently validated this methodology using unstimulated leucaphereses from 5 lymphopenic patients with ALL. PBMCs were activated with anti-CD3/CD28 microbeads (Dynabeads CTS) and then lentivirally transduced with the CAT CAR vector. T cells were then expanded in the WAVE bioreactor before bead removal on a magnetic system and cryopreservation. Patients on study receive lymphodepletion with fludarabine and cyclophosphamide followed by a single dose of 106 CAR+ T cells/kg and are then monitored as an in-patient for 14 days post infusion for toxicities such as cytokine release syndrome or neurotoxicity. The primary end-points of the study are toxicity and the proportion of patients achieving molecular CR at 1 month post CD19CAR T cell infusion. Following this, patients undergo intensive monitoring of disease status for a total of 2 years post infusion. To determine the durability of responses, patients achieving a molecular CR will be monitored closely for the re-emergence of molecular level disease without additional consolidative therapy or SCT Results: We were able to generate the target dose of 1x106 CAR+ T cells/kg in 6 of 7 production runs (involving 2 healthy donors and 5 patients) to date, all of which met sterility release criteria. Transduction efficiency was on average 37% (range 7-84%, see table 1). Mean viral copy was 4.2 (range 1.2-5.8). Memory T cells of stem cell-like phenotype (CAR+ CCR7+ CD45RA+ CD95+ CD127+) formed a mean of 9% (range 0-31%), central memory T cells (CAR+ CCR7+ CD45RA-) formed a mean of 43% (range 16-70%) and effector memory T cells formed a mean of 31% (range 0-77%) of the final CAR T cell product. The percentage of CAR T cells expressing dual exhaustion markers (TIM3+ PD-1+) was on average 5% (range 2-8%). So far 2 patients have been treated. Conclusions We have optimized and successfully validated a robust GMP production method for CD19CAR T cells lentivirally transduced with a novel CD19CAR. Preliminary results of therapy with CAT-41BBz CAR T cells in initial patients on the clinical study will be presented. Disclosures Qasim: Autolus: Consultancy, Equity Ownership, Research Funding; Cellectis: Research Funding; Calimmune: Research Funding; Catapult: Research Funding. Pule:Autolus Ltd: Employment, Equity Ownership, Research Funding; UCL Business: Patents & Royalties; Amgen: Honoraria; Roche: Honoraria.
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Swanson, Jack, Jason Tonne, and Richard Vile. "Activation of APOBEC3B in Self-Inactivating Lentiviral-Based CAR T Cell Production Increases the Mutational Load and Decreases the Killing Efficiency of CAR T Cells." Journal of Immunology 212, no. 1_Supplement (May 1, 2024): 0046_4781. http://dx.doi.org/10.4049/jimmunol.212.supp.0046.4781.
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Abstract Apolipoprotein B mRNA editing enzyme, catalytic polypeptides (APOBECs) are a family of cytidine deaminase enzymes well described in anti-retroviral defense which convert cytidines to uracils (C-U), leading to C-T mutations in DNA. We investigated whether activation of APOBEC3B during the process of making chimeric antigen receptor (CAR) T cells via lentiviral vector affects the overall mutational integrity of resulting CAR T cells. To investigate this question, we quantified APOBEC3B expression and APOBEC signature mutations both post transfection in 293T viral producer cells and post transduction in human T cells. We observed abundant APOBEC3B activation and APOBEC3 signature mutations after transfection of plasmids to make lentivirus, but minimal APOBEC3B activation with a few additional APOBEC3 signature mutations after transduction. To test the functional relevance of APOBEC3B activity, we overexpressed APOBEC3B in 293T cells, and produced lentivirus from the 293T cells overexpressing APOBEC3B. The CAR T cells transduced with lentivirus from the 293T cells overexpressing APOBEC3B exhibited reduced killing ability compared to CAR T cells from 293T cells with no transgene. Taken together, these data suggest that APOBEC3B activation during the process of CAR T cell production induces mutations and reduces CAR T cell killing, and that selective ablation of APOBEC enzymes in GMP-grade lentiviral producer cells may enhance the overall quality of CAR T cells for clinical use.
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Labour, Marie-Noëlle, Camile Le Guilcher, Rachida Aid-Launais, Nour El Samad, Soraya Lanouar, Teresa Simon-Yarza, and Didier Letourneur. "Development of 3D Hepatic Constructs Within Polysaccharide-Based Scaffolds with Tunable Properties." International Journal of Molecular Sciences 21, no. 10 (May 21, 2020): 3644. http://dx.doi.org/10.3390/ijms21103644.
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Organoids production is a key tool for in vitro studies of physiopathological conditions, drug-induced toxicity assays, and for a potential use in regenerative medicine. Hence, it prompted studies on hepatic organoids and liver regeneration. Numerous attempts to produce hepatic constructs had often limited success due to a lack of viability or functionality. Moreover, most products could not be translated for clinical studies. The aim of this study was to develop functional and viable hepatic constructs using a 3D porous scaffold with an adjustable structure, devoid of any animal component, that could also be used as an in vivo implantable system. We used a combination of pharmaceutical grade pullulan and dextran with different porogen formulations to form crosslinked scaffolds with macroporosity ranging from 30 µm to several hundreds of microns. Polysaccharide scaffolds were easy to prepare and to handle, and allowed confocal observations thanks to their transparency. A simple seeding method allowed a rapid impregnation of the scaffolds with HepG2 cells and a homogeneous cell distribution within the scaffolds. Cells were viable over seven days and form spheroids of various geometries and sizes. Cells in 3D express hepatic markers albumin, HNF4α and CYP3A4, start to polarize and were sensitive to acetaminophen in a concentration-dependant manner. Therefore, this study depicts a proof of concept for organoid production in 3D scaffolds that could be prepared under GMP conditions for reliable drug-induced toxicity studies and for liver tissue engineering.
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Bauer, Gerhard, Jon Walker, Bruno Nervi, Julie Ritchey, Jackie Hughes, Bill Eades, Mark Bonyhadi, Jan Nolta, and John F. DiPersio. "Large Scale Ex Vivo GMP Expanded, Activated Human T Cells Consistently Induce Lethal GvHD in a Mouse Xenotransplant Model - A New Way To Study Treatments for Acute GvHD." Blood 106, no. 11 (November 16, 2005): 5242. http://dx.doi.org/10.1182/blood.v106.11.5242.5242.
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Abstract Donor lymphocyte infusions have become increasingly valuable for the treatment of hematologic diseases. In the past it has been reported that ex vivo activated and expanded human (hu) T lymphocytes, compared to fresh, non activated donor lymphocytes, have a markedly reduced ability to elicit normal immune functions in vivo. To overcome this problem, an improved method of ex vivo T cell activation and expansion and a model for in vivo testing of the function of cultured T cells is needed. We developed a more physiological, closed system GMP T cell stimulation protocol using clinical grade magnetic beads coated with CD3 and CD28 antibodies (Xcyte Therapies Inc., Seattle, WA), IL-2 at a low concentration added 24 hours after the initial culture setup and the new GMP grade serumfree Stemline T cell medium (Sigma, St. Louis, MO). This medium allowed the elimination of donor variability, which we experienced in T cell expansions with other serumfree media. Using our NOD SCID/β2m null mouse model of T cell expansion, we demonstrated that high concentrations of IL-2 (500 U/ml, as previously used in clinical T cell expansions) impair the in vivo functionality of expanded T cells. We therefore lowered the IL-2 concentration to 50 U/ml during culture. A 3 fold cell expansion after 4 days, and a 10 fold cell expansion after 8 days of culture could be observed. The CD4 and CD8 ratios were 1.6 ± 0.5 at the start, 1.7 ± 0.4 four days post activation and 1.1 ± 0.2 eight days post-activation, with viability greater than 95%. We conditioned NOD SCID/β2m null mice with 300 cGy of TBI and injected them retroorbitally with 1 x 107 non activated (n=8), 4 day activated (n=9), or 8 day activated hu T cells (n=5). Mice that received 4 or 8 day activated huT cells exhibited weight loss, high hu T cell engraftment, and high hu IFNγ serum levels. All these animals exhibited lethal GvHD with median survivals of 15 and 12 days, respectively. Activated cells outperformed non activated hu T cells in their GvHD potential since not all mice injected with non activated cells developed lethal GvHD. Interestingly, mice which received 4 day activated cells exhibited significantly increased percentages of hu T cells in the blood, spleen, liver and lung, as compared to animals that received non activated hu T cells. With non activated hu T cells, preferential expansion of hu CD4+ cells was seen in mouse organs; with activated hu T cells the CD4/CD8 input ratio was preserved. Although 8 day activated hu T cells also elicited lethal GvHD in all animals, we did not observe the same pattern of engraftment and organ infiltration as with 4 day activated cells. 4 day activated hu T cells produced significantly higher engraftment, hu IFNγ production, and destruction of mouse tissue compared to 8 day activated hu T cells. For the first time, these results demonstrate consistent lethal GvHD elicited in a mouse xenotransplant model by 107ex vivo activated, expanded hu T cells produced in a closed system, serumfree GMP manufacturing process outperforming non activated hu T cells.
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Chang, Sean, Xin Yu, Yongchang Ji, Xiquan Liang, Nektaria Andronikou, and Xavier de Mollerat du Jeu. "Non-viral and viral delivery solutions for next generation cell therapy." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 179.14. http://dx.doi.org/10.4049/jimmunol.200.supp.179.14.
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Abstract The successes of chimeric antigen receptor (CAR) T cells in treating blood cancers have highlighted the cell therapy era. However, the difficulty of delivering molecules into immune cells has been an obstacle to more rapid advancement. Here we present an innovative large-scale Lentivirus (LV) production system as a solution to lower the cost and time of viral production. On the other hand, the next generation cell therapy will rely heavily on gene editing, especially in a safer non-viral integration manner. We have demonstrated that our novel non-viral all-in-one electroporation method provides high efficiency of gene knock-in in primary T cells. The new LV production system was developed for the clinical grade production of lentiviral vectors (LVVs) on a large-scale serum-free suspension platform. This technology employs a newly developed propriety set of GMP reagents comprising of culture media, suspension cells, transfection reagent and boosting enhancers. The system is able to deliver greater than 1 × 108 (TU/mL) functional titer with un-concentrated LVVs. For CRISPR/Cas9 gene editing in primary T cells, we were able to reach more than 90% knockout efficiency for most genes we tested, including T cell receptor (TCR), with Cas9 RNP electroporation using Neon Transfection System. More importantly, gene knock-in efficiency can be reached to greater than 30% with all-in-one electroporation, which delivers Cas9 RNP and donor DNA in one reaction using our newly developed electroporation buffer. Moreover, TCR knockout in addition to a knock-in at another locus can also be done in a single electroporation using our all-in-one method, which is ideal for developing next generation CAR and TCR-T cells.