Lentiviral Vector Production from a Stable Packaging Cell Line Using a Packed Bed Bioreactor

Self-inactivating lentiviral vectors (LVVs) are used regularly for genetic modification of cells, including T cells and hematopoietic stem cells for cellular gene therapy. As vector demand grows, scalable and controllable methods are needed for production. LVVs are typically produced in HEK293T cells in suspension bioreactors using serum-free media or adherent cultures with serum. The iCELLis® is a packed-bed bioreactor for adherent or entrained cells with surface areas from 0.53 to 500 m². Media are pumped through the fixed bed and overflows, creating a thin film that is replenished with oxygen and depleted of CO₂ as media return to the reservoir. We describe the optimization and scale-up of the production of GPRTG-EF1α-hγ_c-OPT LVV using a stable packaging cell line in the iCELLis Nano 2-cm to the 10-cm bed height low compaction bioreactors (0.53 and 2.6 m² surface area) and compare to the productivity and efficacy of GPRTG-EF1α-hγ_c-OPT LVV manufactured under current Good Manufacturing Practice (cGMP) using 10-layer cell factories for the treatment of X-linked severe combined immunodeficiency. By optimizing fetal bovine serum (FBS) concentration, pH post-induction, and day of induction, we attain viral yields of more than 2 × 10⁷ transducing units/mL. We compared transduction efficiency between LVVs produced from the iCELLis Nano and cell factories on healthy, purified CD34⁺ cells and found similar results.