Research paperProduction of HIV virus-like particles by transient transfection of CAP-T cells at bioreactor scale avoiding medium replacement

•CAP-T cells were successfully cultured and transfected in 1 L bioreactor.•VLPs were obtained in bioreactor with the expected quantity and quality.•Medium exchange prior transfection was avoided using a specific media combination.

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 × 106 cells/mL were seeded in 1 L bioreactor. Two days later, ∼2 × 106 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 × 1010 VLPs/mL. This novel strategy significantly simplifies large-scale transient transfection, while suitable cell growth, transfection efficiency, and high quality VLP production are achieved.