Production of HIV virus-like particles by transient transfection of CAP-T cells at bioreactor scale avoiding medium...

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 1L 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, 0.5×10⁶cells/mL were seeded in 1L bioreactor. Two days later, ∼2×10⁶cells/mL were transfected without medium exchange, using 0.5pg of DNA/cell and 3pg of PEI/cell. Transfection efficiency and VLP production comparable to shake flasks were obtained with a production of 4×10¹⁰VLPs/mL. This novel strategy significantly simplifies large-scale transient transfection, while suitable cell growth, transfection efficiency, and high quality VLP production are achieved.