Figure 6. Product quality results from each sampling point across the duration of the run. Black dashed line indicated the acceptable cut-off for the final AEX product. Each datapoint is from a different day in the process. AEX final product sample points are labeled with day of sample collection. (a) HCP titers in ng/mL (acceptable cut-off below line). (b) Percent monomer (acceptable cut-off above line). (c) DNA titers in ng/mL (acceptable cut-off below line). Circled DNA datapoint on AEX step shows out of spec sample due to intentional overloading of AEX membrane.

Advanced Automation Control Strategies

A key objective of this work was to evaluate the ease of integrating multiple unit operations, which requires complex automation strategies. Unit operation integration was handled completely by the PAK system, which removed the burden of in-house automation development and implementation. With the PAK system, unit operation integration is mediated by system-monitored break vessels. For this run, this included the (1) harvest break vessel, (2) protein A capture product vessel, and (3) VI product flow kit vessel. The harvest break vessel and the virus inactivation neutralized product break vessel were controlled to a level setpoint using PID feedback control over the vessel outlet and inlet pump, respectively. The protein A product vessel used minimum, low, high, and maximum level criteria to trigger downstream pause/restart events. Overall, all vessels were controlled as intended. The significance of this control strategy is that it eliminates the concern of over/under flow of intermediate break vessels.
The protein A elution vessel changeover performed on day 9, as described in the Bioburden Control section, resulted in a buildup of cell culture material in the harvest vessel, as the system was paused during this time while perfusion material continued to accumulate. The 10L capacity of the harvest vessel coupled with its 50L overflow bag provided ample time for an operator to swap vessels and restart the system without further incident. The protein A product vessel triggered the downstream operations to begin once reaching a target level of 7.5 liters. After draining to a level of 1.5 liters, the downstream unit operations were triggered to pause to allow the vessel to refill. These trigger levels were manually changed through the HMI throughout the run to allow for the trigger events to coincide with an operator onsite. This was due to an abundance of caution and is not required for the process to operate.
A critical, yet often overlooked parameter in a continuous process is the mass flow rate of the overall process. Rather than set each unit operation to a specified flow rate and over-size break vessels to absorb differences, the PAK system actively controls the flow rate of each individual step and the system as a whole. This is achieved by controlling the last product pump in series to a flow rate set point, while regulating flow of upstream product pumps to maintain intermediate break vessel level (the protein A capture step is an exception). As a result of the vessel level control, any change in flow rate of the last product pump results in a corresponding change in flow rate of the upstream product pumps. This control strategy allows for low break vessel volumes (150 ml for this 50 L cell culture per day process).
The mass flow rate set point (set for the last product pump in series) is determined by the most critical process parameter. For this run, the most critical process parameter was determined to be the virus inactivation residence time. Therefore, the mass flow rate of the entire process was controlled to ensure the VI residence time setpoint was met. A schematic of this process is displayed in Figure 7.