Large-scale Clinical-grade Retroviral Vector Production in a Fixed-Bed Bioreactor

Abstract

The successful genetic engineering of patient T cells with gamma-retroviral vectors expressing chimeric antigen receptors (CARs) or T cell receptors (TCRs) for phase II clinical trials and beyond requires the large-scale manufacture of high titer vector stocks. The production of retroviral vectors from stable packaging cell lines using roller bottles or 10 to 40- layer cell factories is limited by a narrow harvest window, labor intensity, open-system operations, and the requirement for significant incubator space. To circumvent these shortcomings, we optimized the production of vector stocks in a disposable fixed bed bioreactor using GMP-grade packaging cell lines. High titer vector stocks were harvested over 10 days, representing a much broader harvest window than the 3-day harvest afforded by cell factories. For PG13 and 293Vec packaging cells, the average vector titer and the vector stocks’ yield in the bioreactor were higher by 3.2 to 7.3 fold, and 5.6 to 13.1 fold respectively than those obtained in cell factories. The vector production was 10.4 and 18.6 times more efficient than in cell factories for PG13 and 293Vec cells, respectively. Furthermore, the vectors produced from the fixed bed bioreactors passed the release test assays for clinical applications. Therefore, a single vector lot derived from 293Vec is suitable to transduce up to 500 patients cell doses in the context of large clinical trials using CAR- or TCR- T cells. These findings demonstrate for the first time that a robust fixed bed bioreactor process can be used to produce gamma-retroviral vector stocks scalable up to the commercialization phase.

Global Manufacturing of CAR T Cell Therapy

“…Most of this time is spent growing adequate numbers of cells, such as HEK293T cells, to produce large quantities of replication-defective viral vector 39 . Starting from a cryopreserved aliquot of an appropriate working cell bank, the cells are expanded in culture for several days to the appropriate number for production, allowing considerable expansion from the original number of cells seeded.…”

“…The vector systems should employ a number of key safety features that collectively prevent the reacquisition of replication competence (e.g., codon-optimized Gag/Pol that minimizes homology between vector components to prevent recombination, self-inactivating long terminal repeat sequences, and removal of all unnecessary sequences and accessory genes) 41, 42, 43. Within 48 hr of transfection, the production cells begin to release CAR-expressing lentiviral vector, which can be collected from the culture medium 39 . Over the course of several days, medium exchange allows for the harvest of multiple batches of vector-containing medium, typically two harvests.…”

Redirecting T cells with Chimeric Antigen Receptor (CAR) for the treatment of childhood acute lymphoblastic leukemia

“…Despite being a laborious process, construction of RV vectors is highly standardized and the packaging cell lines could be propagated also in Bioreactors, as Pall iCELLis [43,61]. Conversely, the use of LV vectors, which has the advantage to infect also non-dividing cells is still time-consuming and not always economically sustainable [62].…”

Process Development of Adenoviral Vector Production in Fixed Bed Bioreactor: From Bench to Commercial Scale

“…A novel fixed-bed bioreactor, the iCELLis ® provides a recent development providing a polyethylene terephthalate (PET) matrix for adherent cell growth. The iCELLis ® Nano has been used for a range of vector applications, such as for AAV (13), retrovirus (14), Rabies, Hepatitis-A and Chikungunya vaccine production (15). In this study, we evaluated for the first time the fixed bed iCELLis ® bioreactor for the manufacturing of Ad5 vectors in a HEK293 cell line.…”