Figure 7. Schematic of mass flow rate control. (1) FproAeluate: protein A product material flow rate, Facid: acid flow rate, F: total flow into VI SEC column, Fsp: set point flow rate, Eflow: error in target flow into SEC column. (2) Elevel: Error in target vessel level, V: vessel level, Vsp: set point vessel level. (3) Ratio: target ratio of acid flowrate to product flowrate.
The following steps are performed to adjust the pump flow rates to ensure the virus inactivation residence time set point is met. First, the total flow into the virus inactivation SEC column is calculated as the sum of the protein A product feed (VI product pump) and the VI acid pump flow rates. The flow rate set point that corresponds to the virus inactivation residence time target is then subtracted from the total inlet flow, providing the flow rate error. The flow rate error is fed into a PID controller that controls the AEX product pump (the last product pump in series in this process). Because this pump pulls from the VI break vessel, any change in this flow rate will immediately result in a change in VI product vessel level. The error of this vessel is fed into a PID controller that controls the VI product pump to maintain a constant VI vessel level. Finally, the VI acid pump output increases or decreases so that the target ratio between the VI acid and VI product pumps is always achieved.
The virus inactivation residence time for the third VI sub-run is displayed in Figure 5c.
This series of independent control loops, connected only by the physical properties of the VI product vessel allows for cohesive mass flow control across unit operations. Additional unit operations can be added between the VI and AEX steps with this strategy. In this case, flow control cascades up through multiple break vessels.
The perfusion process required a break in mass flow rate at some point in the process. The perfusion rate must be set constant and for this run the VI residence time was controlled to a setpoint. Regardless of the accuracy of pumps, there will inevitably be a difference in mass flow rates between these two unit operations. The break in mass flow rate occurred between the protein A chromatography step and the virus inactivation step. The difference in mass flow rate was absorbed by the protein A product break vessel. This is the reason the protein A product vessel operated under min, low, high, max control rather than continuous PID control and why the virus inactivation step ran as three separate sub batches during this run. In future runs the VI step will operate within a range of acceptable residence times which will allow VI flow rates to adjust to match the changing output of the protein A eluate vessel. This will enable this step to run continuously for the duration of the process instead of periodically stopping and restarting.

Viral Clearance Data from continuous Virus Inactivation

To ensure viral clearance across the flow through virus inactivation SEC column, live virus spiking studies were performed. These studies aimed to (1) characterize the breakthrough profile of virus and (2) to ensure inactivation kinetics occur in the same manner to batch inactivation.
Before testing the column with the virus-spike, the breakthrough profile of the column was characterized with blue dextran as a virus surrogate. Blue dextran is a large molecule with a molecular weight of ~2000kDa (~10-20nm diameter). It is commonly used to determine column void volume and is expected to behave similarly to enveloped viruses, which are typically larger than 50 nm (ICH Q5A, Food and Drug Administration, 1999). Blue dextran spiking studies were performed at various flow rates across the SEC column to characterize the breakthrough profiles of different column residence times. Data can be seen in Figure 8.