Commercial Scale Manufacturing of Allogeneic Cell Therapy

Allogeneic cell therapy products are generating encouraging clinical and pre-clinical results. Pluripotent stem cell (PSC) derived therapies, in particular, have substantial momentum and the potential to serve as treatments for a wide range of indications. Many of these therapies are also expected to have large market sizes and require cell doses of ≥109 cells. As therapeutic technologies mature, it is essential for the cell manufacturing industry to correspondingly develop to adequately support commercial scale production. To that end, there is much that can be learned and adapted from traditional manufacturing fields. In this review, we highlight key areas of allogeneic cell therapy manufacturing, identify current gaps, and discuss strategies for integrating new solutions. It is anticipated that cell therapy scale-up manufacturing solutions will need to generate batches of up to 2,000 L in single-use disposable formats, which constrains selection of currently available upstream hardware. Suitable downstream hardware is even more limited as processing solutions from the biopharmaceutical field are often not compatible with the unique requirements of cell therapy products. The advancement of therapeutic cell manufacturing processes to date has largely been developed with a cell biology driven approach, which is essential in early development. However, for truly robust and standardized production in a maturing field, a highly controlled manufacturing engineering strategy must be employed, with the implementation of automation, process monitoring and control to increase batch consistency and efficiency

Scaling up lentiviral vector production from stable producer cells

Lentiviral vectors (LVs) are commonly used for gene and cell therapies where long-term, sustained expression of therapeutic genes is needed. Legacy methods for LV production include the use of adherent cell lines, transiently transfected with viral packaging genes and the gene of interest (GOI), and cultured in media supplemented with animal sera. However, large scale production is severely limited in adherent cell culture and commercial manufacturing of LV is moving to scalable, serum-free suspension systems. In addition, stable-inducible producer cell lines may eventually replace transient systems for LV production, as this approach circumvents the costs of continually obtaining high quality or cGMP-grade plasmid DNA, the cost of the transfection reagents and the inherent variability of transfection efficiencies. Accordingly, we focused our efforts on workflows that are likely to define the LV manufacturing space in the future and to develop strategies for process development that could enable their uptake and application sooner. For our model, we used an HEK293 derived stable-inducible LV producer cell line (developed by the viral vector production team at the National Research Council Canada in Montreal and described in Manceur et al., 2017) that has been engineered to produce a third generation LV harboring the GFP transgene. A double-inducible system tightly controls the transcription of the envelope glycoprotein VSV-G and the viral Rev genes and allows for normal maintenance and expansion of cultures during seed train development, without loss of viability. This cell line is stable over many passages in culture and no antibiotics were used to maintain a selection pressure. Our goal was to bring the baseline production protocol closer to an industrial workflow that could be closed, scaled and make use of fully chemically defined media and supplements. Optimization of media formulations and feeding regimes at small scales (in shake flasks) led to the development of a multiple harvest, perfusion enabled process in a 1 L stirred tank reactor (STR) as well as a 5-25 L batch process in single use STRs. We demonstrated that to achieve high yields in multiple harvests, cell density needed to be intensified prior to induction and the medium regularly replaced with fresh medium during the production window. To avoid perfusion in the lead up to induction, while reaching a relatively high density of cells (5E6 cells/mL) in the exponential growth phase, we added GE HyClone Cell Boost 5 Supplement (3.5 g/L) to the basal media. After induction, medium was exchanged by continuous perfusion, using an acoustic filter for cell retention, at a rate of one reactor volume per day. High titer (≥1E7 TU/mL) harvests were observed at three, four and five days post induction, including the reactor contents on the fifth day, resulting in four reactor volumes of high titer product at the end of an 11-day process (including the pre-induction culturing time). For higher production scales (5 L and greater) in single use bioreactors, we failed to identify suitable single-use filtering technology that allowed LV to pass freely into the harvest, while retaining the cells in the culture vessel. Therefore, we developed a simple batch process for large scale production that consisted of inoculation, induction and a single harvest at the end of a 6-7-day process (including the pre-induction culturing time). To reach an acceptable volumetric titer (≥1E7 TU/mL) in a batch process, we supplemented the basal medium with GE HyClone LS250 lipid supplement, which resulted in a greater than 3-fold improvement in LV yield over basal media alone. A multiple harvest production mode is higher yielding than a single harvest mode for equivalent culture vessel volumes, however, the single harvest is technically simpler, uses five times less media and supplements, requires less specialized equipment, and has advantages for downstream processing. Therefore, each harvesting mode offers unique advantages and can be used to address specific production needs. In conclusion, we successfully demonstrated a process development path for an industrial workflow for LV manufacturing based on a stable-inducible producer line in fully chemically defined, serum-free media with high volumetric titers. Reference: Manceur, A.P., Kim, H., Misic, V., Andreev, N., Dorion-Thibaudeau, J., Lanthier, S., Bernier, A., Tremblay, S., Gélinas, A.M., Broussau, S., Gilbert, R., Ansorge, S. (2017) Scalable Lentiviral Vector Production Using Stable HEK293SF Producer Cell Lines. Hum Gene Ther Methods. 28 (6):330-339

Development of a closed CAR-T manufacturing process

The field of immunotherapy has emerged as a promising new type of treatment for cancer with the approval of the first two CAR-T therapies. The clinical success of T-cell based immunotherapies necessitates a robust manufacturing process for these products to be consistently produced at commercial scale. Our CAR-T workflow combines unit operation specific solutions for thaw of an apheresis unit, wash, CD3 selection, T-cell activation, lentiviral transduction, incubator- and reactor-based expansion culture, harvest, formulation, cryopreservation and thaw of CAR-T product. We have evaluated the impact of both serum-containing and xeno-free culture media, commercially available T-cell selection and activation reagents, closed small-scale culture vessel options, alternative solutions to enhance transduction, and the specific timing of process steps to develop a modular platform process that is robust and flexible for the varied needs of CAR-T developers. Frozen apheresis units are processed using the SmartWash protocol on the SepaxTM 2 and T-cells are isolated with EasySepTM Release CD3 Positive Selection Kit. The cells are then activated with ImmunoCult CD3/CD28/CD2 T-cell activator before being transduced 24 hours later using the SepaxTM 2. Expansion of Tcells are carried out in two stages: incubator-based culture before going into the XuriTM Cell Expansion System W25 with a perfusion feeding regime. Cultured cells are then harvested and washed in Plasmalyte-A with human serum albumin and formulated with CryoStor® CS10 using the FlexCell protocol on the Sefia™ Cell Processing System. The final cell products are cryopreserved using the VIA Freeze controlled-rate freezer. We have also accessed a point-of-care thawing strategy using the VIA Thaw. Our CAR-T process achieves greater than 1.0E10 expanded T-cells with \u3e80% eGFP transduction efficiency across an 8-day manufacturing process

Industrializing Autologous Adoptive Immunotherapies: Manufacturing Advances and Challenges

Cell therapy has proven to be a burgeoning field of investigation, evidenced by hundreds of clinical trials being conducted worldwide across a variety of cell types and indications. Many cell therapies have been shown to be efficacious in humans, such as modified T-cells and natural killer (NK) cells. Adoptive immunotherapy has shown the most promise in recent years, with particular emphasis on autologous cell sources. Chimeric Antigen Receptor (CAR)-based T-cell therapy targeting CD19-expressing B-cell leukemias has shown remarkable efficacy and reproducibility in numerous clinical trials. Recent marketing approval of Novartis' Kymriah™ (tisagenlecleucel) and Gilead/Kite's Yescarta™ (axicabtagene ciloleucel) by the FDA further underscores both the promise and legwork to be done if manufacturing processes are to become widely accessible. Further work is needed to standardize, automate, close, and scale production to bring down costs and democratize these and other cell therapies. Given the multiple processing steps involved, commercial-scale manufacturing of these therapies necessitates tighter control over process parameters. This focused review highlights some of the most recent advances used in the manufacturing of therapeutic immune cells, with a focus on T-cells. We summarize key unit operations and pain points around current manufacturing solutions. We also review emerging technologies, approaches and reagents used in cell isolation, activation, transduction, expansion, in-process analytics, harvest, cryopreservation and thaw, and conclude with a forward-look at future directions in the manufacture of adoptive immunotherapies