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

Abstract

Large-scale vector manufacturing for phase III and beyond has proven to be challenging. Upscaling the process with suspension cells is increasingly feasible, but many viral production applications are still applicable only in adherent settings. Scaling up the adherent system has proven to be troublesome. The iCELLis(®) disposable fixed-bed bioreactors offer a possible option for viral vector manufacturing in large quantities in an adherent environment. In this study, we have optimized adenovirus serotype 5 manufacturing using iCELLis Nano with a cultivation area up to 4 m(2). HEK293 cell cultivation, infection, and harvest of the virus (by lysing the cells inside the bioreactor) proved possible, reaching total yield of up to 1.6×10(14) viral particles (vp)/batch. The iCELLis 500 is designed to satisfy demand for large-scale requirements. Inoculating a large quantity of cell mass into the iCELLis 500 was achieved by first expanding the cell mass in suspension. Upscaling the process into an iCELLis 500/100 m(2) cultivation area cassette was practical and produced up to 6.1×10(15) vp. Flask productivity per cm(2) in iCELLis Nano and iCELLis 500 was in the same range. As a conclusion, we showed for the first time that iCELLis 500 equipment has provided an effective way to manufacture large batches of adenoviral vectors.

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

“…The same parameters that were previously optimized for iCELLis bioreactor were used for both bioreactor systems. 2,4 Cell growth was found similar in both bioreactors. Productivity in scale-X hydro bioreactor was proven to be at least equally efficient as in iCELLis Nano system.…”

“…Nuclei of cells attached to carriers in iCELLis Nano bioreactor were counted on days 1-4 as previously reported. 2,3 In scale-X hydro bioreactor, there are sampling strips (approximately the same size as carriers in iCELLis Nano bioreactor) located between membrane layers that can be sampled and nuclei counted similar to iCELLis Nano system. 2,3 For each nuclei count, two strips were picked using sterile tweezers.…”

“…2,3 In scale-X hydro bioreactor, there are sampling strips (approximately the same size as carriers in iCELLis Nano bioreactor) located between membrane layers that can be sampled and nuclei counted similar to iCELLis Nano system. 2,3 For each nuclei count, two strips were picked using sterile tweezers. In addition, one scale-X bioreactor fixed-bed was dismantled, and nuclei were counted from top (1 cm from the top edge), middle, and bottom of the bed (1 cm from the bottom), from both membrane layers, and from the outer, middle, and inner surface of the fixed-bed ( Fig.…”

“…Cell lysis was performed 68 h after infection with detergent-based lysis. 2 Harvest material was clarified using 0.027 m 2 DEPTH filters (Millipore, Billerica, MA).…”

“…Our team was one of the first who implemented iCELLis technology for HEK293(T)-adherent cell-based manufacturing process for adenoviral, 2 lentiviral, 3,4 and adeno-associated viral (AAV) 5 vectors. Process development was performed in iCELLis Nano bioreactor, processes were scaled up to iCELLis 500 scale, and currently we have been producing viral vector material in iCELLis 500 for clinical trials.…”