9 research outputs found
Process development of a serum-free and scalable lentiviral vector production in a single-use bioreactor
Lentiviral vectors (LV) have played a critical role in gene delivery for ex vivo gene-modified cell therapies, including T cells and NK cells; however, a large-scale manufacturing process is needed to cope with the increasing demand from the cell and gene therapy sector. Single-use fixed-bed bioreactors, such as the iCELLis bioreactor, provide a promising automated, scalable platform for the generation of large volumes of GMP-grade LV. Fixed-bed bioreactors have already been shown to generate high LV titers; nevertheless, most of the work currently undertaken in the field relies on serum-containing medium formulations. This is largely because to date serum-containing media produces higher viral titer yields and an increased LV stability during the down-stream processing of the product. However, relying on serum-based processes adds significant supply chain and manufacturing risk to the process.
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Process development of a serum-free and scalable lentiviral vector manufacturing platform for cellular immunotherapies
Lentiviral vectors (LV) have played a critical role in gene delivery for ex vivo gene-modified cell therapies, including T cells and NK cells; however, a large-scale manufacturing process is needed to cope with the increasing demand from the cell and gene therapy sector. Single-use fixed-bed bioreactors, such as the iCELLis bioreactor, provide a promising automated, scalable platform for the generation of large volumes of GMP-grade LV. Fixed-bed bioreactors have already been shown to generate high LV titers; nevertheless, most of the work currently undertaken in the field relies on serum-containing medium formulations. This is largely because to date serum-containing media produces higher viral titer yields and an increased LV stability during the down-stream processing of the product. However, relying on serum-based processes adds significant supply chain and manufacturing risk to the process.
Please click Additional Files below to see the full abstract
Process developent of a serum-free and scalable lentiviral vector manufacturing platform for cellular immunotherapies
Lentiviral vectors (LV) have played a critical role in gene delivery for ex vivo gene-modified cell therapies, including T cells and NK cells; however, a large-scale manufacturing process is needed to cope with the increasing demand from the cell and gene therapy sector. Single-use fixed-bed bioreactors, such as the iCELLis bioreactor, provide a promising automated, scalable platform for the generation of large volumes of GMP-grade LV. Fixed-bed bioreactors have already been shown to generate high LV titers; nevertheless, most of the work currently undertaken in the field relies on serum-containing medium formulations. This is largely because to date serum-containing media produces higher viral titer yields and an increased LV stability during the down-stream processing of the product. However, relying on serum-based processes adds significant supply chain and manufacturing risk to the process.
Please click Additional Files below to see the full abstract
Optimizing and developing a scalable, chemically defined, animal component-free lentiviral vector production process in a fixed-bed bioreactor
Lentiviral vectors (LVVs) play a critical role in gene delivery for ex vivo gene-modified cell therapies. However, the lack of scalable LVV production methods and the high cost associated with them may limit their use. In this work, we demonstrate the optimization and development of a scalable, chemically defined, animal component-free LVV production process using adherent human embryonic kidney 293T cells in a fixed-bed bioreactor. The initial studies focused on the optimization of the culture process in 2D static cultures. Process changes such as decreasing cell seeding density on day 0 from 2.5 × 104 to 5 × 103 cells/cm2, delaying the transient transfection from 24 to 120 h post-seeding, reducing plasmid DNA to 167 ng/cm2, and adding 5 mM sodium butyrate 6 h post-transfection improved functional LVV titers by 26.9-fold. The optimized animal component-free production process was then transferred to the iCELLis Nano bioreactor, a fixed-bed bioreactor, where titers of 1.2 × 106 TU/cm2 were achieved when it was operated in perfusion. In this work, comparable functional LVV titers were obtained with FreeStyle 293 Expression medium and the conventional Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum both at small and large scale
Continuous manufacturing of lentiviral vectors
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Continuous production of lentiviral vectors using a fixed-bed bioreactor
Lentiviral vectors (LVs) have emerged as indispensable tools for mediating stable transfer of large transgenes in mammalian cells, which has resulted in the widespread application of LVs for the manufacture of gene-modified cell therapies, particularly chimeric antigen receptor T-cell (CAR-T-cell) therapies.
The typical manufacture of LVs through chemical transfection of adherent embryonic kidney 293T-cells with plasmid DNA is highly versatile and enables manufacturing to pivot to produce vectors with alternative transgenes and envelope proteins. However, transient transfection presents several challenges, including batch-to-batch variability, limited scalability and high costs attributed to the plasmid DNA and transfection reagent. Therefore, the transition towards continuous LV production using producer cell lines would allow for cost effective and scalable manufacturing along with enhanced safety and reproducibility. Specifically, continuous production using stable producer cell lines could extend vector expression by counteracting the limited post-induction expression time and eliminating the requirement for removal of the inducing agent during downstream processing.
In this study, a continuous LV manufacturing process was established using a stable producer cell line, which constitutively expresses third-generation LVs pseudotyped the RDpro envelope protein (WinPacRDpro). Production of LVs in typical 2D culture vessels was performed, however, these systems lack the control of culture parameters, such as pH and dissolved oxygen, and require laborious handling, which introduce risks of contamination. Therefore, the transition to LV production in bioreactors is a necessary step to achieve sufficient LV supply, e.g. for late-stage clinical trials.
This work demonstrates the establishment of a quasi-perfusion process using repetitive batch strategy with medium exchange, first, in 2D culture vessels and then, to an iCELLis fixed-bed bioreactor to continuously produce RDpro-pseudotyped LVs. This initial work using a continuous process-mimic in a fixed-bed bioreactor enables cell expansion in a controlled environment and the bioreactor characterization was used to gain insights for the development of a perfusion strategy. Growth, substrate utilization, lactate generation and LV production were evaluated under different medium exchange strategies. Finally, the established bioreactor process was then transformed to perfusion culture with studies aimed at understanding the impact of perfusion conditions on LV production
Invariant NKT cells as a platform for CAR immunotherapy and prevention of acute Graft-versus-Host Disease
Optimizing and developing a scalable, chemically defined, animal component-free lentiviral vector production process in a fixed-bed bioreactor
Lentiviral vectors (LVVs) play a critical role in gene delivery for ex vivo gene-modified cell therapies. However, the lack of scalable LVV production methods and the high cost associated with them may limit their use. In this work, we demonstrate the optimization and development of a scalable, chemically defined, animal component-free LVV production process using adherent human embryonic kidney 293T cells in a fixed-bed bioreactor. The initial studies focused on the optimization of the culture process in 2D static cultures. Process changes such as decreasing cell seeding density on day 0 from 2.5 × 104 to 5 × 103 cells/cm2, delaying the transient transfection from 24 to 120 h post-seeding, reducing plasmid DNA to 167 ng/cm2, and adding 5 mM sodium butyrate 6 h post-transfection improved functional LVV titers by 26.9-fold. The optimized animal component-free production process was then transferred to the iCELLis Nano bioreactor, a fixed-bed bioreactor, where titers of 1.2 × 106 TU/cm2 were achieved when it was operated in perfusion. In this work, comparable functional LVV titers were obtained with FreeStyle 293 Expression medium and the conventional Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum both at small and large scale