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A rapid increase in the number of gene therapy trials and products has led to a comparable increase in the need for industrial production of viral gene therapy vectors such as lentiviral, adeno-associated, and adenoviral vectors. Current production systems are limited with respect to scalability and robustness. With our CAP® and CAP-T™ cell lines, we have developed a novel system for high-density suspension culture, e ffic ie n t and reproducible transfection, and highly efficient production of viral vectors. By upstream process optimization, we have obtained a robust and high-density fed-batch culture system which can be scaled in any current bioreactor format. A design-of-experiments approach has been employed to optimize transient production of lentiviral vectors with significantly higher titers than can be obtained with adherent HEK293T cells. [ABSTRACT FROM AUTHOR]

Human-derived CAP-T cell line has been demonstrated to be a powerful platform for high-titer production of HIV virus-like particles (VLPs) by PEI-mediated transient transfection. Scale-up of transfection processes is key to ensure the necessary quantities for pre-clinical and clinical testing. One of the major operational challenges of large-scale transient transfection is the medium replacement step that is often required before transfection. In this work, CAP-T cells were cultured in 1 L bioreactor with addition of sodium bicarbonate and surface aeration, which were observed to improve cell state for transfection. Remarkably, the medium replacement step was avoided by culturing the cells in a combination of media (FreeStyleF17 + 1% of PEM) compatible with cell growth and PEI-mediated transient transfection. In the conditions developed in this work, 0.5 × 10 6 cells/mL were seeded in 1 L bioreactor. Two days later, ∼2 × 10 6 cells/mL were transfected without medium exchange, using 0.5 pg of DNA/cell and 3 pg of PEI/cell. Transfection efficiency and VLP production comparable to shake flasks were obtained with a production of 4 × 10 10 VLPs/mL. This novel strategy significantly simplifies large-scale transient transfection, while suitable cell growth, transfection efficiency, and high quality VLP production are achieved. [ABSTRACT FROM AUTHOR]