Removal of empty capsids from adeno-associated virus (AAV) manufacturing lots remains a critical step in the downstream processing of AAV clinical-grade batches. Because of similar physico-chemical characteristics, the AAV capsid populations totally lacking or containing partial viral DNA are difficult to separate from the desired vector capsid populations. Based on minute differences in density, ultracentrifugation remains the most effective separation method and has been extensively used at small scale but has limitations associated with availabilities and operational complexities in large-scale processing. In this paper, we report a scalable, robust, and versatile anion-exchange chromatography (AEX) method for removing empty capsids and subsequent enrichment of vectors of AAV serotypes 5, 6, 8, and 9. On average, AEX resulted in about 9-fold enrichment of AAV5 in a single step containing 80% ± 5% genome-containing vector capsids, as verified and quantified by analytical ultracentrifugation. The optimized process was further validated using AAV6, AAV8, and AAV9, resulting in over 90% vector enrichment. The AEX process showed comparable results not only for vectors with different transgenes of different sizes but also for AEX runs under different geometries of chromatographic media. The herein-reported sulfate-salt-based AEX process can be adapted to different AAV serotypes by appropriately adjusting elution conditions to achieve enriched vector preparations.

Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19 + B-cell malignancies in numerous clinical trials. The CAR molecule, which recognizes cell-surface tumor-associated antigen independently of human leukocyte antigen (HLA), is composed by one or more signaling molecules to activate genetically modified T cells for killing, proliferation, and cytokine production. In order to make this treatment available for a larger number of patients, we developed a simple and efficient platform to generate and expand CAR-T cells. Our approach is based on a lentiviral vector composed by a second-generation CAR that signals through a 41BB and CD3-ζ endodomain. In this work, we show a high-level production of the lentiviral vector, which was successfully used to generate CAR-T cells. The CAR-T cells produced were highly cytotoxic and specific against CD19+ cells in vitro and in vivo, being able to fully control disease progression in a xenograft B-cell lymphoma mouse model. Our work demonstrates the feasibility of producing CAR-T cells in an academic context and can serve as a paradigm for similar institutions. Nevertheless, the results presented may contribute favoring the translation of the research to the clinical practice.