Induced pluripotent stem cells (iPSCs) have emerged as a consistent cell source for ‘off-the-shelf’ therapeutic T cells, with particular interest in CD8 T cells for CAR-T cell applications. ...Traditionally, Notch signaling, crucial for T-lineage differentiation, has been delivered through DLL4-expressing feeder cells or DLL4 protein-coated vessels in static culture, but these methods have proven inefficient for generating CD8 T cells from iPSCs at therapeutic scale. We have previously shown a method involving our proprietary DLL4/VCAM-1-conjugated magnetic beads (‘Engineered Thymic Niche’; ETN) for precisely modulating Notch signaling in a scalable suspension bioreactor-based culture. In this study, we have used ETN technology for the generation of functional CD8+ T cells in a stirred tank bioreactor, starting with a clonal iPSC cell line with a CD19-CAR inserted in at the TRAC locus. The CD8 T cells were functionally capable of multiple rounds of in vitro tumor cell lysis and sustained tumor growth inhibition in vivo.
iPSCs expressing CD19-CAR were differentiated to CD34+ cells in 250 mL stirred-tank reactors (STR), yielding an average of 300 million cells per batch, at 87% CD34+ purity and an efficiency of 3.5 lympho-competent progenitors per input expanded iPSC. iPSC-derived CD34+ cells were further differentiated in a 250mL STR in the presence of ETN, yielding over 5 x 106 mature CD4-CD8+ T cells mL-1 expressing engineered CAR with high efficiency. This ETN-based cell differentiation process has shown to be readily scalable with Proof-of-Concept (PoC) of up to 1.6L STR showing similar outputs to 250mL.
CD19-CAR expressing iPSC-CD8+ T cells derived using the scalable STR platform were capable of multiple rounds of in vitro cytotoxic activity to a range of effector cells (E:T ratio) proliferation, and cytokine secretion comparable to primary T cells. Furthermore, tumor growth inhibition potential of STR-derived iPSC-CD8+ T cells in an in vivo PoC study, using a mouse model with CD19+ tumor, was comparable to primary T cells. This is a significant advancement in generation of ‘off-the-shelf’, highly functional CD8+ T cells using a clonal iPSC line in a small footprint automated bioreactor which have greater capacity for linear scale-up and process control than traditional T cell manufacturing systems. This PoC study is a step toward the development of robust, cost-effective, safe, and efficacious allogeneic immunotherapies.
Despite their continued clinical success, the manufacturing of autologous chimeric antigen receptor T cell (CAR-T) therapies remains prohibitively expensive. The emergence of next-generation ...allogeneic products, designed for universal patient administration, promises substantial reductions in manufacturing costs if economies of scale can be achieved. To meet this potential, robust bioprocesses in scalable manufacturing platforms that can support high cell yields and quality are urgently required. The aim of this work was therefore to optimise and establish a multi-litre, perfusion stirred-tank bioreactor (STR) CAR-T manufacturing workflow poised for future allogeneic production.
A Design of Experiments (DOE) study was first conducted in the Ambr® 250 to investigate the impact of alternating tangential flow (ATF) perfusion parameters, and donor variability on anti-CD19 CAR-T cell growth and quality. Identifying optimal perfusion conditions in serum-free media supported final CAR-T cell yields almost five times greater than the conventional fed-batch process (Figure 1). Harvested CAR-T cells exhibited functional characteristics, minimal exhaustion, and predominantly a desirable naïve and central memory phenotype. Optimised perfusion settings were subsequently scaled-up into the Univessel® Single-Use (SU) 2L STR and comparability across both scales was assessed. Initial scale-up experiments conducted in fed-batch mode have confirmed comparability between the Ambr® 250 and Univessel® SU 2L in terms of cell growth kinetics (Figure 2). The integration of the Ksep® closed system for automated cell harvesting was also characterised and found not to adversely impact cell phenotype nor viability. These studies showcase the substantial improvements in CAR-T yields achievable through data-driven optimisation of perfusion parameters in STRs. Additionally, they establish a scalable multi-litre STR workflow suited for future allogeneic CAR-T manufacturing needs.
A new biomanufacturing platform combining intracellular metabolic engineering of the oleaginous yeast Yarrowia lipolytica and extracellular bioreaction engineering provides efficient bioconversion of ...plant oils/animal fats into high‐value products. However, predicting the hydrodynamics and mass transfer parameters is difficult due to the high agitation and sparging required to create dispersed oil droplets in an aqueous medium for efficient yeast fermentation. In the current study, commercial computational fluid dynamic (CFD) solver Ansys CFX coupled with the MUSIG model first predicts two‐phase system (oil/water and air/water) mixing dynamics and their particle size distributions. Then, a three‐phase model (oil, air, and water) utilizing dispersed air bubbles and a polydispersed oil phase was implemented to explore fermenter mixing, gas dispersion efficiency, and volumetric mass transfer coefficient estimations (kLa). The study analyzed the effect of the impeller type, agitation speed, and power input on the tank's flow field and revealed that upward‐pumping pitched blade impellers (PBI) in the top two positions (compared to Rushton‐type) provided advantageous oil phase homogeneity and similar estimated kLa values with reduced power. These results show good agreement with the experimental mixing and kLa data.
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Yarrowia lipolytica has shown to be a promising biomanufacturing platform for efficient bioconversion of plant oils/animal fats into high‐value products. In this CFD study, the authors demonstrate three‐phase CFD models predicting the hydrodynamics and mass transfer parameters of efficient yeast fermenters with high agitation and sparging required for well‐dispersed oil droplets in an aqueous medium. This work demonstrates the utility of CFD for predicting and scaling these critical efficient fermentation parameters.
Advanced cell and gene therapies such as chimeric antigen receptor T‐cell immunotherapies (CAR‐T), present a novel therapeutic modality for the treatment of acute and chronic conditions including ...acute lymphoblastic leukemia and non‐Hodgkin lymphoma. However, the development of such immunotherapies requires the manufacture of large numbers of T‐cells, which remains a major translational and commercial bottleneck due to the manual, small‐scale, and often static culturing systems used for their production. Such systems are used because there is an unsubstantiated concern that primary T‐cells are shear sensitive, or prefer static conditions, and therefore do not grow as effectively in more scalable, agitated systems, such as stirred‐tank bioreactors, as compared with T‐flasks and culture bags. In this study, we demonstrate that not only T‐cells can be cultivated in an automated stirred‐tank bioreactor system (ambr® 250), but that their growth is consistently and significantly better than that in T‐flask static culture, with equivalent cell quality. Moreover, we demonstrate that at progressively higher agitation rates over the range studied here, and thereby, higher specific power inputs (P/M W kg−1), the higher the final viable T‐cell density; that is, a cell density of 4.65 ± 0.24 × 106 viable cells ml−1 obtained at the highest P/M of 74 × 10−4 W kg−1 in comparison with 0.91 ± 0.07 × 106 viable cells ml−1 at the lowest P/M of 3.1 × 10−4 W kg−1. We posit that this improvement is due to the inability at the lower agitation rates to effectively suspend the Dynabeads®, which are required to activate the T‐cells; and that contact between them is improved at the higher agitation rates. Importantly, from the data obtained, there is no indication that T‐cells prefer being grown under static conditions or are sensitive to fluid dynamic stresses within a stirred‐tank bioreactor system at the agitation speeds investigated. Indeed, the opposite has proven to be the case, whereby, the cells grow better under higher agitation speeds while maintaining their quality. This study is the first demonstration of primary T‐cell ex vivo manufacture activated by Dynabeads® in an automated stirred‐tank bioreactor system such as the ambr® 250 and the findings have the potential to be applied to multiple other cell candidates for advanced therapy applications.
This study focuses on the expansion of primary human T‐cells isolated from healthy donors in stirred tank bioreactors (ambr 250). Results show that higher stirring speed and higher specific power input do not have an impact on the final product composition and also lead to a higher final cell yield.
Hepatocyte‐like cells derived from human‐induced pluripotent stem cells (hiPSC‐HLC) are expected to have important applications in drug screening and regenerative medicine. However, hiPSC‐HLC are ...difficult to produce on a large‐scale to obtain relevant numbers for such applications. The aim of this study was to implement a novel integrated strategy for scalable production of hiPSC‐HLC and demonstrate the applicability of dielectric spectroscopy to monitor hiPSC expansion/differentiation processes. We cultured hiPSC as three‐dimensional (3D) aggregates in stirred‐tank bioreactors (STB) operated in perfusion with an in situ capacitance probe. Dissolved oxygen concentration and dilution rate were controlled along the process and after 5 days of cell expansion, the hepatic differentiation was integrated in sequential steps for 28 days. The hiPSC were able to grow as 3D aggregates and the expression of hepatic markers and albumin production after differentiation confirmed that hepatocyte differentiation improved when compared to 2D culture. These hiPSC‐HLC exhibited functional characteristics of hepatocytes including glycogen storage and drug metabolization capacity. Our results also show a good correlation between the cell permittivity measured online and the aggregate biovolume measured by standard offline methods, demonstrating for the first time the potential of dielectric spectroscopy to monitor hiPSC expansion and differentiation in STB.
A novel integrated bioprocess for expansion and hepatic differentiation of human induced pluripotent stem cells (hiPSC) as 3D aggregates was developed in this study using scalable stirred‐tank bioreactors operated in perfusion. The authors also show for the first time the potential of dielectric spectroscopy as a process analytical technology for in situ monitoring of hiPSC growth and differentiation status in 3D cultures.
Produced water (PW) and crude glycerin (CG) are compounds overproduced by the oil and biodiesel industry and significant scientific efforts are being applied for properly recycling them. The aim of ...this research is to combine such industrial byproducts for sustaining the production of xanthan by Xanthomonas campestris. Xanthan yields and viscosity on distinct PW ratios (0, 10, 15, 25, 50, 100) and on 100% dialyzed PW (DPW) in shaker batch testing identified DPW treatment as the best approach for further bioreactor experiments. Such experiments showed a xanthan yield of 17.3 g/L within 54 h and a viscosity of 512 mPa s. Physical‐chemical characterization (energy dispersive X‐ray spectroscopy, scanning electron microscopy and Raman spectroscopy) showed similarities between the produced gum and the experimental control. This research shows a clear alternative for upcycling high salinity PW and CG for the generation of a valued bioproduct for the oil industry.
Chimeric antigen receptor T‐cell (CAR‐T) therapies have proven clinical efficacy for the treatment of hematological malignancies. However, CAR‐T cell therapies are prohibitively expensive to ...manufacture. The authors demonstrate the manufacture of human CAR‐T cells from multiple donors in an automated stirred‐tank bioreactor. The authors successfully produced functional human CAR‐T cells from multiple donors under dynamic conditions in a stirred‐tank bioreactor, resulting in overall cell yields which were significantly better than in static T‐flask culture. At agitation speeds of 200 rpm and greater (up to 500 rpm), the CAR‐T cells are able to proliferate effectively, reaching viable cell densities of >5 × 106 cells ml‐1 over 7 days. This is comparable with current expansion systems and significantly better than static expansion platforms (T‐flasks and gas‐permeable culture bags). Importantly, engineered T‐cells post‐expansion retained expression of the CAR gene and retained their cytolytic function even when grown at the highest agitation intensity. This proves that power inputs used in this study do not affect cell efficacy to target and kill the leukemia cells. This is the first demonstration of human CAR‐T cell manufacture in stirred‐tank bioreactors and the findings present significant implications and opportunities for larger‐scale allogeneic CAR‐T production.
Chimeric antigen receptor T‐cell therapies (CAR‐T) have proven clinical efficacy for the treatment of hematological malignancies, however have high manufacturing costs. The authors demonstrate expansion of CAR‐T cells in stirred‐tank bioreactors and found that T‐cells grow better under agitated conditions whilst maintaining final product quality and function. Successful expansion of CAR‐T cells in stirred‐tank bioreactors provides opportunities for larger‐scale allogeneic therapies.
A continuous Chinese hamster ovary (CHO) cell culture process comprised of a highly proliferative N‐1 perfusion bioreactor utilizing a hydrocyclone as a cell retention device linked to a production ...continuous‐flow stirred tank reactor (CSTR) is presented. The overflow stream from the hydrocyclone, which is only partially depleted of cells, provides a continuous source of high viability cells from the N‐1 perfusion bioreactor to the 5–20 times larger CSTR. Under steady‐state conditions, this linked‐bioreactor system achieved a peak volumetric productivity of 0.96 g/L/day, twofold higher than the optimized fed‐batch process. The linked bioreactor system using a hydrocyclone was also shown to be 1.8–3.1 times more productive than a dual, cascading CSTR system without cell retention.
Hydrocyclones are small and simple devices that alleviate fouling and scale‐up concerns associated with conventional perfusion methodologies. We have demonstrated an N‐1 perfusion bioreactor utilizing a hydrocyclone for cell retention linked to a 5–20× larger production continuous‐flow stirred tank reactor operating at a steady state. This linked bioreactor system reaches 2× the volumetric productivity of the optimized fed‐batch process while enabling the use of legacy manufacturing infrastructure up to 10,000 L.
Mesenchymal stem cells (MSCs) from the Wharton’s jelly (WJ), a mucoid connective tissue of the umbilical cord having several therapeutic advantages are implicated in numerous clinical trials with ...target indications ranging from hematological to neurodevelopmental disorders. The effective cellular dose of WJ-MSCs derived from tissues is at a very low quantity, for application in pre-clinical/clinical trials; thus further commercialization relies on biomanufacturing to retain its proliferative multipotent state and therapeutic integrity. We have optimized the large-scale production of GMP-compliant xeno-free/serum-free human WJ-MSCs using a microcarrier-based stirred-tank bioreactor (5 L). A total yield of 2 × 109 (2.3 ± 400.34 ×109) cells/batch at a concentration of 5.0 × 105 cells/mL was achieved using a microcarrier platform in 14.0 ± 1.73 days corresponding to a 20 ± 1 fold expansion and harvest efficiency of 98.3 ± 4.5%. The upscaled WJ-MSCs were characterized and found to be of standard clinical grade. WJ-MSC inhibited phytohemagglutinin (PHA) mediated T-cell proliferation in vitro, the inhibitory potential could be enhanced by cytokine preconditioning of cells. A decrease in both the CD4 + and CD8 + populations and concomitant reduction in the CD25 + activation marker was noted.
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•Large-scale production of human Wharton’s jelly (WJ) derived mesenchymal stem cells (MSCs) was achieved.•The microcarrier based stirred-tank bioreactor produced xeno-free/serum-free clinical grade cells.•A total yield of 2 × 109 (2.3 ± 400.34 ×109)/batch at a concentration of 5.0 × 105 cells/mL was achieved.•The cells had consistent batch to batch characteristics and the process is GMP-compliant.•The scaled-up cells have high demand and potential application in inflammatory/immunomodulatory clinical conditions.
The rapid depletion of crude-oil resource which sustains a conventional petroleum refinery together with its environmental impact has led to the search for more sustainable alternatives. In this ...context, biorefinery serves to fulfil the aim by utilizing waste resources. Hence, this study focused on techno-economic assessment of PHB production at large scale from waste carob pods in a closed-loop biorefinery setup. Firstly, the use of pure sugars in SC1 was shifted to use of carob pods as feedstock in SC2, upgradation of stirred tank bioreactor with novel annular gap bioreactor in SC3 and replacing the conventional centrifugation process with the upcoming ceramic membrane separation process in SC4. An Aspen plus™ flowsheet was developed by including the aforementioned novel strategies for PHB production. The effectiveness of PHB production under various scenarios was evaluated based on its pay-out period and turnover accumulated at the end of 7th year of a PHB plant operation. Instead of pure sugars as the feedstock (SC1), carob pod extract (SC2) reduced the pay-out period from 12.6 to 6.8 years. Likewise, switching onto ABR from the conventional STBR further decreased the pay-out period to 4.8 years. Finally, the use of ceramic membranes (SC4) instead of centrifugation resulted in a similar pay-out period of 4.8 years with increased turnover of about 1.4 billion USD. Thus, the use of carob pods along with an improved PHB titre in ABR and incorporation of affordable ceramic membrane technology for PHB rich biomass separation resulted in a highly cost-effective PHB production strategy.
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•Aspen Plus flowsheet for PHB production with novel bioprocessing strategies is presented.•Utilization of carob pods as feedstock reduced pay-out period from 12.6 to 6.8 years.•Replacement of STBR with ABR reduced pay-out period from 6.8 to 4.8 years.•Use of membrane separation resulted in an annual turnover of 1.4 billion USD.
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