Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection

Abstract

The need for γ-retroviral (gRV) vectors with a self-inactivating (SIN) design for clinical application has prompted a shift in methodology of vector manufacturing from the traditional use of stable producer lines to transient transfection-based techniques. Herein, we set out to define and optimize a scalable manufacturing process for the production of gRV vectors using transfection in a closed-system bioreactor in compliance with current good manufacturing practices (cGMP). The process was based on transient transfection of 293T cells on Fibra-Cel disks in the Wave Bioreactor. Cells were harvested from tissue culture flasks and transferred to the bioreactor containing Fibra-Cel in the presence of vector plasmid, packaging plasmids and calcium-phosphate in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Virus supernatant was harvested at 10–14 h intervals. Using optimized procedures, a total of five ecotropic cGMP-grade gRV vectors were produced (9 liters each) with titers up to 3.6×107 infectious units per milliliter on 3T3 cells. One GMP preparation of vector-like particles was also produced. These results describe an optimized process for the generation of SIN viral vectors by transfection using a disposable platform that allows for the generation of clinical-grade viral vectors without the need for cleaning validation in a cost-effective manner.

Critical Variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency

“…16,17 After transfection, the media was changed to X-Vivo 10 and vector was harvested at 12 h intervals. During process development, vector transfected in DMEM in 10-layer CellSTACKs and vector transfected in X-Vivo 10 on Fibra-Cel carriers showed comparable titer (11.7 × 10 5 versus 12.6 × 10 5 IU ml −1 , respectively; Figure 1a).…”

“…16,17 Briefly, a mixture of plasmid DNA and 0.25 M CaCl 2 (Sigma-Aldrich) was added drop wise to an equal volume of 2X HEPES-buffered saline, pH 7.05 (Sigma-Aldrich), while slowly bubbling air through the HEPES-buffered saline solution. For transfection in tissue culture plastic (T-75 or CellSTACKs), optimal amounts of plasmid DNA were 10 μg of vector, 9 μg of gag/pol and 4 μg of envelope in 10 ml of media per 75 cm 2 of surface area.…”

“…17 However, during scale-up for the clinical production of the IL2RG vector, which was to be produced in serum-free X-Vivo media as opposed to the serum-containing media used in our original report, 17 we encountered new problems. An investigation led to the identification of several additional factors that affected production of high titer virus using large-scale transient-transfection-based methodologies.…”

Manufacture of tumor- and virus-specific T lymphocytes for adoptive cell therapies

“…In the case of gamma retroviral vectors with a self-inactivating design, vector manufacturing relies on transient transfection-based techniques similar to lentiviral vector production. 70 …”

“…The available scalable expansion systems include the cell factory system, the HYPERFlask, microcarriers and bioreactors. 70,94,95 The downstream processes for lentiviral vector production aims at removing cell and plasmid contaminants, concentrating vector particles to achieve high titer vector stocks while maintaining vector potency. These are challenging tasks that typically encompass the following steps: (1) Vector stocks harvesting.…”