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Background & Aim
Lentiviral vectors (LVV) are the most common delivery method for transducing T cells for CAR-T therapies, and their production and purification are major cost-drivers in manufacturing; an industry “gold standard” of 10-20% recovery results in oversized and expensive production batches. While methods for purifying monoclonal antibodies and vaccines are well established as platform processes, effective and consistent purification strategies for LVV are a serious challenge due to 1) the labile nature of the virus, 2) physically segregating LVV from the cells from which they bud, 3) removing host cell DNA and protein, and 4) 0.2 µm sterile filtration for 0.08-0.12 µm particles at high concentrations. An ideal purification platform would solve these problems and handle material from adherent and suspension-based cells, as well as stable producers and a range of transient-transfection conditions in a closed, scalable manner. Although unit operations from mAB and vaccine bioprocessing are readily available, they have yet to be successfully commercialized for LVV (e.g. affinity chromatography) or are detrimental to infectivity (e.g. anion exchange). Furthermore, membrane/resin chemistries and consumable sizes have been singly optimized for bioprocessing. Thus, a host of wildly divergent, open, and non-scalable schemes are being developed across industry and academia (e.g. spin filters vs. TFF vs. AEX), resulting in poor recoveries, inconsistency of product, and risk of contamination.
Methods, Results & Conclusion
At GE Fast Trak Toronto/CCRM we are developing a closed, single-use, ambient temperature, single-day downstream lentiviral vector purification process that aims to robustly and consistently purify and concentrate LVV to minimums of 1E8 TU/mL and 25% recovery with 2-3 log reduction of host cell protein and DNA. I will discuss development and current progress of taking a chimeric approach to downstream processing by aiming to replace non-scalable ultracentrifugation with depth filtration (mABs), non-existent affinity chromatography with TFF (biologics), applying a novel multimodal chromatography for purification (vaccines), and developing effective methods for sterile filtration, all in a closed manner with maximum automation. The combination of single-use consumables, reduced processing time, and increased recovery will greatly reduce COGs for the entire CAR-T manufacturing process and thus increase access to these powerful new treatments.
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© 2019 Published by Elsevier Inc.