Production of recombinant adeno-associated vectors using two bioreactor configurations at different scales

Abstract

The conventional methods for producing recombinant adeno-associated virus (rAAV) rely on transient transfection of adherent mammalian cells. To gain acceptance and achieve current good manufacturing process (cGMP) compliance, clinical grade rAAV production process should have the following qualities: simplicity, consistency, cost effectiveness, and scalability. Currently, the only viable method for producing rAAV in large-scale, e.g. > or =10(16) particles per production run, utilizes baculovirus expression vectors (BEVs) and insect cells suspension cultures. The previously described rAAV production in 40 L culture using a stirred tank bioreactor requires special conditions for implementation and operation not available in all laboratories. Alternatives to producing rAAV in stirred tank bioreactors are single-use, disposable bioreactors, e.g. Wave. The disposable bags are purchased pre-sterilized thereby eliminating the need for end-user sterilization and also avoiding cleaning steps between production runs thus facilitating the production process. In this study, rAAV production in stirred tank and Wave bioreactors was compared. The working volumes were 10 L and 40 L for the stirred tank bioreactors and 5 L and 20 L for the Wave bioreactors. Comparable yields of rAAV, approximately 2E+13 particles per liter of cell culture were obtained in all volumes and configurations. These results demonstrate that producing rAAV in large scale using BEVs is reproducible, scalable, and independent of the bioreactor configuration.

Fig. 1

Distribution of rAAV capsid proteins (VP1, VP2 and VP3) analyzed by A) SDS-PAGE; and B) Western Blot. Only fractions with a density close to 1.41 g/cm³ of the CsCl gradient at 72hpi from each bioreactor configuration were analyzed. Lane 1) Low molecular weight marker; 2) Western blot marker. Densities in g/cm³ of the CsCl fractions for 3) 200ml, 1.4075; 4) 5L Wave, 1.4307; 5) 20 L Wave, 1.4128; 6) 10 L STB, 1.4180; 7) 40 L STB1, 1.4328; 8) 40L STB 2, 1.4159 and 9) 40L STB 3, 1.4117.

Fig. 1

Distribution of rAAV capsid proteins (VP1, VP2 and VP3) analyzed by A) SDS-PAGE; and B) Western Blot. Only fractions with a density close to 1.41 g/cm³ of the CsCl gradient at 72hpi from each bioreactor configuration were analyzed. Lane 1) Low molecular weight marker; 2) Western blot marker. Densities in g/cm³ of the CsCl fractions for 3) 200ml, 1.4075; 4) 5L Wave, 1.4307; 5) 20 L Wave, 1.4128; 6) 10 L STB, 1.4180; 7) 40 L STB1, 1.4328; 8) 40L STB 2, 1.4159 and 9) 40L STB 3, 1.4117.

Fig. 2

Yield of vector genomes of rAAV produced in different bioreactor configurations and scales. The yields correspond to 1L of cell culture harvested at 72hpi. The vg titers were obtained by qPCR.

Fig. 3

Yield of transducing units of rAAV produced in different bioreactor configurations and scales. The yields correspond to 1L of cell culture harvested at 72hpi. The tu titers were obtained by transducing HEK-293 cells.

Fig. 4

Ratio of tu/vg for the rAAV produced in different configuration of bioreactors and scales.

Fig. 5

Distribution of vg (●) and tu (■) across the CsCl gradient from rAAV produced in A) shake flask; B) 20L wave bioreactor and C) 40L stirred tank bioreactor at 72 hpi. The vg titer was obtained by qPCR and the tu by transduction assay in HEK-293 cells.

Fig. 5

Distribution of vg (●) and tu (■) across the CsCl gradient from rAAV produced in A) shake flask; B) 20L wave bioreactor and C) 40L stirred tank bioreactor at 72 hpi. The vg titer was obtained by qPCR and the tu by transduction assay in HEK-293 cells.

Fig. 5

Distribution of vg (●) and tu (■) across the CsCl gradient from rAAV produced in A) shake flask; B) 20L wave bioreactor and C) 40L stirred tank bioreactor at 72 hpi. The vg titer was obtained by qPCR and the tu by transduction assay in HEK-293 cells.