Tu157 - A Scalable and Robust Method to Produce and Obtain Pure Lentiviral Vectors for Point-of-care CART-cell Therapy Developed in Academic Settings

Abstract Text: The Hospital Clinic de Barcelona (HCB) is a European leader in developing CART-cells targeting CD19 for B-cell malignancies. Indeed, its (Varnimcabtagene autoleucel, Var-cel) therapy for relapsed/refractory acute lymphoblastic leukemia has been approved for hospital exemption use and received PRIME designation by the EMA. Var-cel, as other CAR-T therapies, relies on lentiviral vectors (LVVs) to deliver transgenes to autologous T cells. Thus, optimizing and scaling LVVs production is crucial to meet growing demand and regulatory standards.
We developed an optimized and scalable LVV production pipeline at HCB using a fixed-bed bioreactor (Scale-X Hydro) achieving a threefold increase in LVV productivity compared to our conventional, currently-approved, upstream production (USP) using 2D multi-layer flasks (~6x10⁶ versus ~2x10⁶ TU/cm²). The revised USP was based on prior optimizing of 293T cell growth and transfection conditions. Further downstream processing (DSP) was also refined to yield high LVV purity with minimal loss of functional titer. DSP steps included DNAse treatment, clarification via filtration, concentration/diafiltration using hollow fiber filters and fast protein liquid chromatography with multimodal resin, which enabled the removal of over 99% of process-related impurities while retaining a high LVV functional titer (~2.5x10⁸ TU/ml) and a recovery rate exceeding 60%.
Our optimized LVVs production and purification pipeline aligns with industry standards and supports efforts to secure EMA marketing authorization, ensuring scalable LVV supply for point-of-care CAR-T therapies.