Purpose of ReviewRecent developments in regenerative medicine have precipitated the need to expand gene-modified human T cells to numbers that exceed the capacity of well-plate-based, and flask-based processes. This review discusses the changes in process development that are needed to meet the cell expansion requirements by utilizing hollow-fiber bioreactors. Maintenance of cell proliferation over long periods can become limited by unfilled demands for nutrients and oxygen and by the accumulation of waste products in the local environment.Recent FindingsPerfusion feeding, improved gas exchange, and the efficient removal of lactate can increase the yield of T cells from an average of 10.8E +09 to more than 28E +09 in only 10 days.SummaryAggressively feeding cells and actively keeping cells in the bioreactor improves gas exchange and metabolite management over semi-static methods. The ability to remove the environmental constraints that can limit cell expansion by using a two-chamber hollow-fiber bioreactor will be discussed.

Lentiviral vectors are widely used in the field of gene therapy as an effective method for permanent gene delivery. While current methods of producing small scale vector batches for research purposes depend largely on culture flasks, the emergence and popularity of lentiviral vectors in translational, preclinical and clinical research has demanded their production on a much larger scale, a task that can be difficult to manage with the numbers of producer cell culture flasks required for large volumes of vector. To generate a large scale, partially closed system method for the manufacturing of clinical grade lentiviral vector suitable for the generation of induced pluripotent stem cells (iPSCs), we developed a method employing a hollow fiber bioreactor traditionally used for cell expansion. We have demonstrated the growth, transfection, and vector-producing capability of 293T producer cells in this system. Vector particle RNA titers after subsequent vector concentration yielded values comparable to lentiviral iPSC induction vector batches produced using traditional culture methods in 225 cm2 flasks (T225s) and in 10-layer cell factories (CF10s), while yielding a volume nearly 145 times larger than the yield from a T225 flask and nearly three times larger than the yield from a CF10. Employing a closed system hollow fiber bioreactor for vector production offers the possibility of manufacturing large quantities of gene therapy vector while minimizing reagent usage, equipment footprint, and open system manipulation.

Cell-based therapies hold great promise for a myriad of clinical applications. However, as these therapies move from phase I to phase II and III trials, there is a need to improve scale-up of adherent cells for the production of larger good manufacturing practice (GMP) cell banks. As we advanced our neural stem cell (NSC)-mediated gene therapy trials for glioma to include dose escalation and multiple treatment cycles, GMP production using cell factories (CellStacks) generated insufficient neural stem cell (NSC) yields. To increase yield, we developed an expansion method using the hollow fiber quantum cell expansion (QCE) system. Seeding of 5.2 × 107 NSCs in a single unit yielded up to 3 × 109 cells within 10 days. These QCE NSCs showed genetic and functional stability equivalent to those expanded by conventional flask-based methods. We then expanded the NSCs in 7 units simultaneously to generate a pooled GMP-grade NSC clinical lot of more than 1.5 × 1010 cells in only 9 days versus 8 × 109 over 6 weeks in CellStacks. We also adenovirally transduced our NSCs within the QCE. We found the QCE system enabled rapid cell expansion and increased yield while maintaining cell properties and reducing process time, labor, and costs with improved efficiency and reproducibility.

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With the recent successes of CAR-T cell therapies there has been a renewed interest in bioprocessing and the manufacturing facilities required to meet the demands. Increasing the scale of any process has potential risks as well as rewards. While the drive to automation is clear, there is a lot of debate on the strategy. The decision on whether and when to automate a particular step can have long-term consequences on the therapies' cost-of-goods (COGs) and their successes. Automating the most complex and error-prone steps of a process is thought to be the best alternative to manual processes. However, automation may not be possible or even recommended for all steps in a process. Here, we examine both the barriers and the advantages of automation. Additionally, a detailed discussion of automating the autologous CAR-T cell process will look at each step from collection to infusion, and discuss the current automation offerings and speculate on potential future process changes. Through application of process analytics, we can gain a deeper understanding of the processes' consistency and tailor automation to fit the process. New potency assays will improve manufacturing processes and ensure the delivery of a safe and effective product. These advancements will then shape the cell therapy laboratory of our future.