Academic literature on the topic 'GMP-Clinical Grade Processing'

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.

Abstract Abstract 4316 Therapeutic applications of T cells in immunotherapy have recently gained momentum with the promising results in adoptive transfer of antigen-specific T cells for infectious complications after allogeneic stem cell or solid organ transplantation or for immunotherapy of malignant diseases. Activation and expansion of these cells for clinical application under controlled conditions require GMP-grade reagents including appropriate antibodies, cytokines and media. For standardized, reproducible cell cultivation and ex vivo differentiation procedures, a new serum and xeno-component free, GMP-grade medium for clinical use has been developed: High lot-to-lot consistency has been achieved by eliminating protein components not relevant for T cell expansion leaving human serum albumin as the only protein component. The expansion of T cells in this medium upon polyclonal activation using biotinylated antibodies against CD2, CD3 and CD28 loaded on anti-Biotin coated beads resulted in expansion rates similar to other commercially available serum-free media. Using soluble antibodies against CD3 and CD28, more than 30%-higher expansion rates of viable and functional T cells after 6 days of expansion have been achieved with the new xeno-component free medium compared with other serum-fee media. Transferring the same protocol to a high density cell culture system such as a gas permeable rapid expansion device, high densities of T cells with more than 1.5×107 cells/ mL were reached. The generation of antigen-specific T cells using the Cytokine Capture System (IFN-gamma) and the serum and xeno-component free T cell medium showed similar results regarding purity, recovery and background stimulation compared to the use of a standard basal medium supplemented with 10% human AB serum. For the automation of such complex procedures, a new cell processing device was developed. All steps for the antigen-specific T cell processing, i.e. antigen-specific re-stimulation, magnetic enrichment, and in vitro expansion with this T cell medium are performed in this fully automated device, in a closed system under sterile conditions. In conclusion, the newly developed GMP-grade, serum and xeno-component free T cell medium demonstrated high lot-to-lot consistency and was superior in its performance to other commercially available serum-free media in high density cell culture systems. The new medium can be used to replace human AB serum supplementation for the clinical manufacturing of T cells resulting in easier handling and higher consistency. Disclosures: Assenmacher: Miltenyi Biotec GmbH: Employment. Mockel-Tenbrinck:Miltenyi Biotec GmbH: Employment. Scheffold:Miltenyi Biotec GmbH: Employment. Rauser:Miltenyi Biotec GmbH: Employment. Bohnenkamp:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec GmbH: Employment. Odenthal:Miltenyi Biotec GmbH: Employment. Veit:Miltenyi Biotec GmbH: Employment. Kolrep:Miltenyi Biotec GmbH: Employment. Fahrendorff:Miltenyi Biotec GmbH: Employment.

Abstract Generation and characterization of unrestricted somatic stem cells (USSC) from cord blood (CB) was described by our group and has been well established under laboratory conditions [Koegler et al 2004, 2005 and 2006; Sensken et al 2007]. Due to their proliferative and differentiation capacity, USSCs are an interesting candidate for the future development of cellular therapy for tissue repair and tissue regeneration as well as a supportive cell layer to support hematopoietic reconstitution. Since generation and expansion under GMP-grade conditions is mandatory for use in clinical application, the automated cell processing system Sepax (BIOSAFE) with the CS900 separation kit was used for mononuclear cell separation and the subsequent generation of the USSC colonies in the presence of 30% GMP-grade fetal calf serum (Perbio), low-glucose DMEM-medium/10-7M dexamethasone. Expansion of USSC was performed in a closed system (Macopharma) applying cell stacks (Costar Corning). Results achieved so far indicate that the generation frequency and quality of generated USSC under GMP conditions are equal or even superior (45%) to manual generation under laboratory conditions (43%). 20 cord-blood units (mean volume 88,5 +− 15,8 ml; mean number of mononuclear cells 3,1 +−0,6 *108 MNC) have been processed, resulting in 9 USSC-colony formations and lines within 14–28 days. Growth kinetics is equal to the previously established USSC-lines (∼36–48 h / population doubling). Analysis of the immunophenotype as well as the differentiation potential towards the mesenchymal, neural and endodermal lineages also showed no difference to those lines generated manually using Ficoll-separation and normal cell culture flasks (Costar Corning T225). The closed system applied here is perfectly suitable to ensure safe and easy handling of the USSC, including seeding, trypsination and harvesting. In combination with the cell stack system (1, 2, 5 and 10 layers), cell amounts of more than 1.0×109 USSC can be achieved within 4 passages. These USSC products were temperature controlled cryopreserved in the presence of 10% DMSO, HSA and dextran. USSC can be thawed and further expanded in clinical grade quality. On the basis of their pluripotency and expansion under GMP-conditions into large quantities, these USSC from cord blood, when pretested for infectious agents and matched for the major transplantation antigens, may serve as a universal allogeneic stem cell source for tissue repair and tissue regeneration.

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.

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

e22080 Background: Early phase clinical trials of investigational agents benefit from laboratory assays that quantify intended mechanism of action on molecular target (1° PD effects), desired changes in mechanistic biomarkers (2° PD effects), and 3° PD effects on cell survival. Robust PD assay results are valuable for informing go/no‐go decisions about continued development of new agents and for identifying combinations of targeted agents that cover the multiple molecular defects underlying many malignancies. Methods: Readying PD assays for clinical use involves validating analytical performance, identifying and qualifying critical reagents, demonstrating fit‐for‐purpose for the clinical protocol, and finalizing companion SOPs specifying specimen handling and processing. Because clinical PD questions often demand assay performance that meets or even exceeds clinical diagnostic assay standards, but key assay reagents are usually R and D‐rather than GMP‐grade, stringent reagent Quality Control is critical for preventing assay failures due to lot-to-lot variability. Results: The poly(ADP-ribose) (PAR) Immunoassay was the first qualified assay developed by NCI for implementation in early phase clinical trials. Development of stringent production and internal Quality Control specifications for accepting/rejecting new lots of critical reagents and a defined Quality Assurance Plan allowed a network of users to achieve consistent results and quality using R&D-grade source materials. Conclusions: The NCI’s Division of Cancer Treatment and Diagnosis is developing a clinical PD assay portfolio capable of quantifying 1°, 2°, and 3° PD effects. Proven clinical assays are formally transferred from the NCI to requesting sites in academia and industry via laboratory‐based certification and training, centralized access to SOPs, assistance with assay transfer, and participation in the assay’s Quality Assurance Plan. Funded by NCI Contract No HHSN261200800001E.

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

Abstract Reactivation of CMV remains a major cause of morbidity and mortality in immunocompromised recipients of allogeneic stem cell transplantation. Antiviral pharmacotherapy may not be sufficient due to significant toxicity and moderate efficacy. It has been shown that adoptive transfer of donor-derived CMV-specific T cells may be an effective strategy to control established CMV infection. For a persistent function in vivo the presence of both virus-specific CD8+ and CD4+ T cells is essential. Therefore, we developed an optimized protocol for the generation of CMV pp65-specific CD8+ and CD4+ T cell lines which is fully compliable with Good Manufacturing Practice (GMP) conditions. Enrichment for CMV-specific T cells followed by only a short culture period is likely to retain maximal in vivo potential. PBMCs from 7 CMV seropositive donors were stimulated with recombinant pp65 protein (7–70 μg/ml) and/or HLA-A*0201/HLA-B*0702 restricted immunodominant pp65 peptides (NLV/TPR). Peptides used were clinical grade, and recombinant protein was gamma-irradiated (50 kGy, −80 C°) to eliminate possible microbiological contamination. High dose gamma-irradiation of pp65 protein resulted in partial degradation, but antigenic presentation was maintained. IFNγ producing cells were enriched using the IFNγ secretion assay (Miltenyi Biotec) at day 1 after stimulation, and cultured with autologous feeders (10x) and IL-2 (10 or 50 IU IL-2/ml) with or without CD3/28 expansion beads. Addition of high concentrations of protein during initial stimulation had a negative effect on enrichment probably due to non-specific stimulation of cells. Addition of immunodominant pp65 peptides promoted isolation efficiency and proliferation of epitope-specific CD8+ T cells in some donors. Cell lines were analyzed at different time points (day 4–15) using peptide-MHC tetramer and phenotypic markers. In addition, pp65-specificity was evaluated by intracellular IFNγ staining after restimulation with a pp65 protein-spanning pool of 15-mer peptides. CMV-specific lysis was tested in a 51-Cr release assay on pp65-transduced target cells. Enrichment of IFNγ producing cells after pp65 protein stimulation resulted in pp65-specific cell lines consisting of both CD8+ and CD4+ T cells. The T cell subset distribution directly after enrichment did not change during culture and was reproducible for each donor. Moreover, the composition of T cell lines reflected the pp65-specific response in donor PBMC starting material. The CD8+ compartment contained the known immunodominant tetramer staining cells (range 5–100%). The majority of both CD8+ and CD4+ T cells produced IFNγ upon restimulation with the pp65 peptide-pool, and showed CMV-specific lysis of target cells. The phenotype of pp65-specific T cells was predominant CD28+/CD45RO+ and CD45RA−/CCR7−/CD62L−, although CCR7 and CD62L were transiently expressed at day 4 and 7 after stimulation. Cryopreservation did not affect the composition or functionality of T cell lines. In conclusion, this procedure yields GMP-grade T cell lines comprising both CD8+ and CD4+ CMV-specific T cells. Processing and presentation of CMV protein by donor antigen-presenting cells enables selection of the full pp65-specific donor repertoire, without restrictions related to HLA or known epitopes. The choice for a moderate or more vigorous expansion after enrichment remains arbitrary and needs to be evaluated in clinical trials.

Introduction: The manufacturing process for CD19-directed CAR T cells, a life-saving therapy for B-ALL, remains first generation: non-automated, labor-intensive, and requiring expensive clean room facilities for GMP cell manufacture. This approach impacts both patient (pt) safety and access, producing constraints on availability of therapy and manufacturing turnaround. Use of the CliniMACS Prodigy platform allows for GMP, rapid, semi-automated, clinical-scale processing of humanized (hu)CART19 cell products in a point-of-care system and addresses many of these constraints. Based on preclinical data that demonstrated superior efficacy of huCART19 T cells manufactured on the Prodigy platform, we launched a Phase 1/2 study (NCT 05480449) of huCART19 for pediatric B-ALL, with complete Phase 1 and interim Phase 2 results presented here. Methods: Phase 1 included only children and young adults previously treated with CAR T cells. Phase 2 included both CAR-naïve and CAR-exposed patients (pts). After fludarabine/cyclophosphamide lymphodepletion, pts were infused with Prodigy-manufactured huCART19 cells. Results: From 11/2022-05/2024, 17 pts were enrolled. Of 16 pts infused (median age 12 years, range 3-26), 11 were CAR-exposed and 5 were CAR-naïve. Among CAR-exposed pts, 6 had relapsed after CAR therapy, and 5 had developed early B cell recovery. Of CAR-naïve pts, 2 had primary refractory and 3 had multiply-relapsed (all post hematopoietic stem cell transplant, HSCT) disease. Ten pts had evidence of disease at infusion: morphologic (n=6), non-CNS extramedullary (n=1), measurable residual disease by flow cytometry (flow-MRD, n=1), or by high-throughput sequencing (NGS-MRD, n=2). 100% of pts had cells successfully manufactured at the protocol dose in 7-9 days. The median infused cell dose was 4.6x106 huCART19 cells/kg (range, 2-5x106). For all but one patient, 3 surplus doses were also manufactured. Marginal manufacturing cost per patient was $28,427. Dose optimization occurred without a dose limiting toxicity (DLT), and the recommended phase 2 dose (RP2D) was achieved at 5x106 huCART19 cells/kg. Cytokine release syndrome (CRS) was observed in 11 pts (grade 1=8, grade 2=2, grade 3=1) and immune effector cell-associated neurotoxicity syndrome (ICANS) in 2 pts (grade 3=1, grade 4=1). In the Phase 2 retreatment setting, 1 pt with a prior history of seizure disorder and severe neurotoxicity after prior CAR therapy developed fatal cerebral edema in the setting of grade 4 ICANS on day 7. Among the 15 pts evaluable for response at day 28, the overall response rate was 100% (complete morphologic remission, n=14; partial response, n=1 [patient with non-CNS extramedullary disease]), 14 of whom (93%) were flow-MRD negative. There were 13 pts evaluable for NGS (Clonoseq)-MRD at day 28, of which 10 (77%) had undetectable NGS-MRD. One additional pt cleared NGS-MRD at month 3 without intervention. In CAR-naïve pts, 5/5 pts (100%) achieved flow MRD-negative CR; although, 1 patient remained with detectable NGS-MRD. In CAR-exposed pts evaluated at day 28, flow MRD-negative CR was achieved in 8/10 (80%) with one pt with detectable NGS-MRD at day 28 that cleared by month 3 without intervention. With a median follow-up of 4.7 months, event-free survival at 6 months was 94% (95% CI 83%-100%) and at 12 months was 78% (95% CI 53%-100%); pts were censored at alternate treatment or HSCT (n=2) and study withdrawal (n=1). There have been no additional patient deaths from disease or toxicity. Conclusion: Prodigy-manufactured huCART19 T cells yielded a 100% overall response rate in evaluable pts, with a 100% MRD-negative CR rate in CAR-naïve patients, and an 80% flow MRD-negative CR rate in patients with a poor response to prior CAR. This trial shows feasibility of manufacturing 4-1BB cells on the Prodigy cost-effectively, with successful manufacture for each pt and all products meeting release criteria. Investigation will continue into the significance of NGS-MRD positivity following Prodigy-manufactured huCART19 therapy, durability of remission, and potentially faster automated GMP manufacturing.

e14009 Background: Dendritic cells (DCs) are one of the central tools in cellular anti-tumor immunotherapy, being characterized by their capacity for acquiring and processing antigens and ability to produce strong antitumor immune responses. The production of clinical grade ex-vivo monocyte-derived DCs (Mo-DCs) is the most frequent approach for antitumor vaccines production. Recently, therapeutic resistance to radio/chemotherapy and disease recurrence was shown to be in part due to a small cancer stem cell (CSCs) population present in tumors. Methods: Here, we aim to target and eradicate CSCs by developing a novel DC-based immunotherapy vaccine for pancreatic and non-small cells lung cancer (NSCLC), comparing the loading of CSCs vs. classical tumor lysates. Results: CSCs from PANC-1 (pancreatic cancer) and A549 (NSCLC) cell lines were successfully isolated and characterized, overexpressing stem-like markers: NANOG, OCT4, SOX2 and CD133. CSCs resistance to Gemcitabine was also assessed. Before comparing the 2 types of vaccine loading, we also analyzed the impact of 3 GMP free-serum culture media on the phenotype and functional abilities of Mo-DCs. DCs cultured in X-VIVO 15 and AIM-V media show enhanced production of IL-12 and are able to induce a superior stimulation of T cells, mainly CTLs and Th1 subsets. By contrast, DCs cultured in DendriMACS are more prone to induce Treg polarization. Conclusions: Overall, our data demonstrate that blood monocytic precursors present considerable plasticity allowing a tailored differentiation of DCs just by changing the nutritive support. We also highlight the need of critically defining the culture medium to be used in DC cancer immunotherapy in order to attain desired cell characteristics and by consequent robust responses. Finally, our preliminary results indicate that loading DCs with CSCs antigens may be an effective strategy to target and destroy this resilient cancer cell population.

L'optimisation de la purification des cellules d'îlots est essentielle pour faire progresser les thérapies cellulaires ciblant le diabète de type 1, nécessitant l'intégration de technologies innovantes, conformes aux normes GMP, afin de renforcer l'efficacité des processus, l'automatisation et l'évolutivité. Cette thèse analyse deux technologies majeures employées dans la purification des îlots. La première étude examine l'impact d'un nouveau système de refroidissement sur le processus de purification des cellules d'îlots. Ce système permet un contrôle précis de la température durant la purification par gradient de densité grâce à un refroidissement sous pression, stabilisant efficacement la température tout en préservant la stérilité de l'environnement de salle blanche GMP. Comme solution rentable et peu invasive, ce système de refroidissement présente également des applications potentielles pour d'autres équipements de thérapie cellulaire, dont la majorité est dépourvue d'option de refroidissement. Le principal axe de cette thèse porte sur l’adaptation du système Sepax C Pro - Sefia, une technologie en circuit fermé initialement conçue pour le traitement des cellules souches hématopoïétiques, à la purification des cellules d'îlots humains. Avec le retrait progressif du système Cobe 2991 en Europe d'ici 2025 et mondialement d'ici 2031, la plateforme Sepax C Pro - Sefia constitue une alternative automatisée et conforme aux normes GMP. Elle intègre l’automatisation des étapes critiques du processus de purification des îlots, réduit la manipulation manuelle et améliore la reproductibilité des processus, en s'imposant comme une solution innovante pour les applications cliniques et de recherche en transplantation d'îlots. À la suite de ces avancées, un brevet intitulé « Systèmes et Méthodes pour le Traitement des Tissus » a été déposé pour protéger la méthode innovante développée pour le système reconverti Sepax C Pro - Sefia, assurant ainsi la sécurisation de la propriété intellectuelle et facilitant son application future en milieu clinique. Grâce à l'intégration de ces innovations technologiques, y compris une méthode brevetée pour le traitement des tissus, cette thèse propose un cadre exhaustif pour le remplacement du système Cobe 2991, garantissant la continuité de l'isolement clinique des îlots et contribuant à des thérapies plus efficaces pour les patients atteints de diabète de type 1 (allogreffes), ainsi que pour ceux souffrant de pathologies pancréatiques (autogreffes)
The optimization of islet cell purification is crucial for advancing cell-based therapies for type 1 diabetes, requiring innovative, GMP-compliant technologies to improve process efficiency, automation, and scalability. This thesis evaluates two key technologies used in islet purification. The first study evaluates the impact of a novel cooling system on the islet cell purification process. This system ensures precise temperature control during density gradient purification by providing pressurized cooling, effectively stabilizing the temperature while maintains the sterility of the GMP cleanroom environment. As a cost-effective and minimally invasive solution, this cooling system holds promises to cool another cell therapy equipment the majority of which offer no cooling option.The core focus of My thesis is the repurposing of the Sepax 2C Pro- Sefia system, a closed-system technology originally developed for hematopoietic stem cell processing, for human islet cell purification. With the Cobe 2991 system being phased out in Europe by 2025 and globally by 2031, the Sepax 2 - Sefia platform offers a fully automated, GMP-compliant alternative. It automates key steps in the islet purification process, reduces manual handling, and improves process reproducibility, making it a ground-breaking solution for both clinical and research applications in islet transplantation. Building on the findings a patent: “Systems and Methods for Tissue Processing,” was filed to protect the novel approach developed for the repurposed Sepax 2 - Sefia system ensuring the intellectual property is secured and facilitating the future application of this system in clinical settings. Through the integration of these technological advancements, including a patented method for tissue processing, this thesis provides a comprehensive framework to replace the Cobe 2991 system, ensuring the continuity of clinical islet isolation and contributing to more effective therapies for patients with type1 diabetes (allografts) but also patients with pancreatic pathologies (autografts)