Development and Optimization of AAV hFIX Particles by Transient Transfection in an iCELLis^®Fixed-Bed Bioreactor

Abstract

Adeno-associated virus (AAV) vectors are increasingly popular in gene therapy because they are unassociated with human disease, replication dependent, and less immunogenic than other viral vectors and can infect a variety of cell types. These vectors have been used in over 130 clinical trials, and one AAV product has been approved for treatment of lipoprotein lipase deficiency in Europe. To meet the demand for the increasing quantities of AAV required for clinical trials and treatment, a scalable high-capacity technology is required. Bioreactors meet these requirements but limited options are available for adherent HEK 293T/17 cells. Here we optimize the transient transfection of HEK293T/17 cells for the production of AAV human factor IX in a disposable fixed-bed bioreactor, the iCELLis(®) Nano (PALL Corporation). A fixed bed in the center of the iCELLis bioreactor is surrounded by culture medium that is pumped through the bed from the bottom of the bioreactor so that a thin film of the medium overflows the bed and is replenished with oxygen and depleted of CO2 as it returns to the surrounding medium reservoir. We show that this fixed-bed bioreactor can support as many as 2.5 × 10(8) cells/ml of fixed bed (1.9 × 10(6) cells/cm(2)). By optimizing culture and transfection parameters such as the concentration of DNA for transfection, day of harvest, size of PEI/DNA particles, and transfection medium, and adding an additional medium change to the process, we increased our yield to as high as 9.0 × 10(14) viral particles per square meter of fixed bed. We also show an average GFP transfection of 97% of cells throughout the fixed bed. These yields make the iCELLis a promising scalable technology for the clinical production of AAV gene therapy products.

Benchmarking of Scale-X Bioreactor System in Lentiviral and Adenoviral Vector Production

“…However, for sure, for the production of other viral vectors, such as AAV, larger bioreactors are needed, as the required viral vector numbers per patient are often higher compared to lentiviral vectors. 8,37,38 Adenoviral vector production in iCELLis Nano and scale-X bioreactors…”

“…6 We have been able to produce more than 1 • 10 16 adenoviral particles per batch. Others have also found iCELLis bioreactor useful for retrovirus, 7 AAV, 8 Rabies, 9 Hepatitis-A, 9 and Chikungunya 9 vaccines, or for recombinant protein production in insect cells. 10 iCELLis 500 bioreactor is good manufacturing practice (GMP) compliant, fully disposable, and controlled system with perfusion capability.…”

Manufacturing of recombinant adeno‐associated viruses using mammalian expression platforms

“…Cell disruption or breakage at large‐scale is achieved either by microfluidizer or detergents (e.g. Triton X‐100 at 0.1 to 0.5%) . As mentioned under small‐scale purification, for convenience, nuclease is added during or after the cell breakage to reduce the DNA/RNA content and the viscosity of the lysate for subsequent steps.…”

Optimization of lentiviral vector production for scale-up in fixed-bed bioreactor

“… 19 , 20 iCELLis allows scaling of adherent production in a controlled environment, and the highly integrated single-use equipment can be adapted to meet current good manufacturing practices requirements. Viral vaccines, 21 recombinant proteins, 22 adeno-associated viral vectors 23 and retroviral vectors 16 have been produced in iCELLis Nano. In addition, adenoviral vector production has been scaled-up to iCELLis 500.…”