Enabling Allogeneic T Cell-Based Therapies: Scalable Stirred-Tank Bioreactor Mediated Manufacturing.

Allogeneic T cells are key immune therapeutic cells to fight cancer and other clinical indications. High T cell dose per patient and increasing patient numbers result in clinical demand for a large number of allogeneic T cells. This necessitates a manufacturing platform that can be scaled up while retaining cell quality. Here we present a closed and scalable platform for T cell manufacturing to meet clinical demand. Upstream manufacturing steps of T cell activation and expansion are done in-vessel, in a stirred-tank bioreactor. T cell selection, which is necessary for CAR-T-based therapy, is done in the bioreactor itself, thus maintaining optimal culture conditions through the selection step. Platform's attributes of automation and performing the steps of T cell activation, expansion, and selection in-vessel, greatly contribute to enhancing process control, cell quality, and to the reduction of manual labor and contamination risk. In addition, the viability of integrating a closed, automated, downstream process of cell concentration, is demonstrated. The presented T cell manufacturing platform has scale-up capabilities while preserving key factors of cell quality and process control.

Scale-up manufacturing of gelatin-based microcarriers for cell therapy.

Microcarriers, including crosslinked porous gelatin beads (Cultispher G) are widely used as cell carriers for cell therapy applications. Microcarriers can support a range of adherent cell types in stirred tank bioreactor culture, which is scalable up to several thousands of liters. Cultispher G in particular is advantageous for cell therapy applications because it can be dissolved enzymatically, and thus cells can be harvested without the need to perform a large-scale cell-bead filtration step. This enzymatic dissolution, however, is challenged by the slow degradation of the carriers in the presence of enzymes as new extracellular matrix is being deposited by the proliferating cells. This extended dissolution timelimits the yield of cell recovery while compromising cellular viability. We report herein the development of crosslinked porous gelatin beads that afford rapid, stimuli-triggered dissolution for facile cell removal using human mesenchymal stem cells (hMSC) as a model system. We successfully fabricated redox-sensitive beads (RS beads) and studied their cell growth, dissolution time and cell yield, compared to regular gelatin-based beads (Reg beads). We have shown that RS beads allow for much faster dissolution compared to Reg beads, supporting better hMSC detachment and recovery following 8 days of culture in spinner flasks, or in 3L bioreactors. These newly synthesized RS beads show promise as cellular microcarriers and can be used for scale-up manufacturing of different cell types while providing on-demand degradation for facile cell retrieval.

End-to-End Platform for Human Pluripotent Stem Cell Manufacturing.

Industrialization of stem-cell based therapies requires innovative solutions to close the gap between research and commercialization. Scalable cell production platforms are needed to reliably deliver the cell quantities needed during the various stages of development and commercial supply. Human pluripotent stem cells (hPSCs) are a key source material for generating therapeutic cell types. We have developed a closed, automated and scalable stirred tank bioreactor platform, capable of sustaining high fold expansion of hPSCs. Such a platform could facilitate the in-process monitoring and integration of online monitoring systems, leading to significantly reduced labor requirements and contamination risk. hPSCs are expanded in a controlled bioreactor using perfused xeno-free media. Cell harvest and concentration are performed in closed steps. The hPSCs can be cryopreserved to generate a bank of cells, or further processed as needed. Cryopreserved cells can be thawed into a two-dimensional (2D) tissue culture platform or a three-dimensional (3D) bioreactor to initiate a new expansion phase, or be differentiated to the clinically relevant cell type. The expanded hPSCs express hPSC-specific markers, have a normal karyotype and the ability to differentiate to the cells of the three germ layers. This end-to-end platform allows a large scale expansion of high quality hPSCs that can support the required cell demand for various clinical indications.

Platforms for Manufacturing Allogeneic, Autologous and iPSC Cell Therapy Products: An Industry Perspective.

As cell therapy processes mature from benchtop research protocols to industrial processes capable of manufacturing market-relevant numbers of doses, new cell manufacturing platforms are required. Here we give an overview of the platforms and technologies currently available to manufacture allogeneic cell products, such as mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), and technologies for mass production of autologous cell therapies via scale-out. These technologies include bioreactors, microcarriers, cell separation and cryopreservation equipment, molecular biology tools for iPSC generation, and single-use controlled-environment systems for autologous cell production. These platforms address the challenges of manufacturing cell products in greater numbers while maintaining process robustness and product quality.

Bioavailability, rheology and sensory evaluation of fat-free yogurt enriched with VD3 encapsulated in re-assembled casein micelles.

Vitamin D3 (VD3) deficiency is a global problem. Better ways are needed to enrich foods with this important nutraceutical. VD3 is fat-soluble, hence requiring a suitable vehicle for enriching nonfat foods. Our objectives were to assess the bioavailability of VD3, from fat-free yogurt, in re-assembled casein micelles (rCMs) compared to that in polysorbate-80 (PS80/Tween-80) a commonly used synthetic emulsifier, and to assess and compare their rheology and palatability. We enriched fat-free yogurt with VD3 loaded into either rCM (VD3-rCMs) or PS80 (VD3-PS80). In vivo VD3 bioavailability was evaluated by a large randomized, double blind, placebo-controlled clinical trial, measuring serum 25(OH)D increase in subjects who consumed fat-free yogurt with 50,000 IU of either VD3-rCM, VD3-PS80, or VD3-free placebo yogurt. Both VD3-rCM and VD3-PS80 increased the serum 25(OH)D levels by ∼8 ng ml(-1) and no significant differences in mean 25(OH)D levels were observed, evidencing the fact that VD3 bioavailability in rCM was as high as that in the synthetic emulsifier. VD3-rCM yogurt had a higher viscosity than VD3-PS80 yogurt. In sensory evaluations, panelists were able to discern between VD3-rCM and VD3-PS80 yogurt, and showed a dislike for PS80 yogurt, compared to rCM or the unenriched control. These results complement our past results showing higher protection against thermal treatment, UV irradiation, and deterioration during shelf life, conferred to hydrophobic nutraceuticals by rCM compared to that by the synthetic surfactant or to the unprotected bioactive, in showing the advantageous use of rCM over the synthetic emulsifier as a delivery system for the enrichment of food with VD3 and other hydrophobic nutraceuticals.