Two-stage cultivation systems used for semi-continuous and continuous MVA virus production.

A) Two-stage semi-continuous cultivation (SSC) system for small-scale MVA production using shaker flasks. Cells were produced in semi-continuous mode in the Small Cell Bioreactor (SCB; working volume 120 mL) and transferred to the Small Virus Bioreactor (SVB; 65, 120 or 200 mL working volume), where virus infection and propagation took place. Twice a day, a semi-continuous harvest was taken (V₄), cells were transferred from SCB to SVB (V₂), and fresh medium was added to SCB and SVB (V₁ and V₃). The volumes of harvest, cell transfer and fresh medium were determined with Eqs 1–4 (see Materials and Methods). Shakers without baffles were used. B) Two-stage continuous stirred tank bioreactor (TSB, 1 L Sartorius Biostat B plus; the working volume of CB and VB was 850 and 440 mL, respectively) system using AGE1.CR.pIX cells with a production flow rate (F₄) of 0.29 mL/min. Continuous cell production was maintained in the first bioreactor (Cell Bioreactor, CB). Cells were continuously transferred at a flow rate (F₂) of 0.18 mL/min to a second vessel (Virus Bioreactor, VB; dilution rate 0.0390 h^-1), where MVA virus infection and propagation took place. Fresh medium was added to CB and VB at a flow rate of 0.18 (F₁) and 0.11 mL/min (F₃), respectively.