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Background Chimeric Antigen Receptor (CAR) T cell manufacturing is associated with high costs. LipidBrick® Cell Ready is a novel lipid-based nanoparticle solution that can be easily loaded with mRNA and DNA payloads for transfecting T cells, without the need for specialized instruments.Methods Peripheral blood derived T cells were seeded into stirred bioreactor and activated. MODDE ®’s Design of Experiments (DOE) methodology was used to optimize the generation of T cell receptor (TCR) knockout (KO) T cells using the LipidBrick® Cell Ready lipid particles in static transfections.Results Activation induced robust T cell proliferation and stirring had no impact on cell growth. DOE guided optimization of KO efficiencies and T cell expansion identified optimal setpoints. Using these setpoints, TCR KO efficiencies >80% (pre-purification) were reproducibly observed across multiple donors, while maintaining cell growth.Conclusions Through DOE-guided optimization, we efficiently manufactured TCR KO T cells using significantly lower RNA to what is typically required in electroporation based-processes, thus simplifying manufacture and mitigating costs.Ethics Approval This study was approved by Sartorius’s Institutional Biosafety Committee (IBC). Project No.: 2024-02. Project title: T cell process development.