Figure 8: Principle of a flow rate gradient for optimization of productivity and resin utilization. (A) set-up of sensors in the chromatographic workstation (B) change of flow rate vs time or volume loaded onto the column and breakthrough curves at low and high residence time blue line high residence time, redline low residence time, (C) resin utilization productivity and yield of an unoptimized process and a process with model predictive control, data from (Eslami et al., 2022a; Eslami et al., 2022b).
The system can be also used for continuous chromatography. Then two columns are operated in tandem, while one column is loaded and the second column is washed, eluted and regenerated. If the wash, elution, and regeneration cycle is longer than loading then four columns must be installed. The selection of column number follows the same rules as for counter current chromatography. Furthermore, the velocity gradient approach can be extended to wash, elution and regeneration and thereby even higher productivity could be obtained. During loading in counter current chromatography two columns are interconnected and therefore the maximal system pressure and flowrate is constrained by this situation. This is not the case when columns are operated in parallel with velocity gradients during loading.
How fully integrated continuous biomanufacturing may look like was been previously proposed by the Morbidelli group (Feidl et al., 2020). They propose a process wide control of the integrated process. The unit operations are individually controlled and process wide by a supervisory system. The challenge is to integrate all unit operations and to maintain a constant mass flow and to avoid propagation of process errors or deviations. Therefore, always surge tanks between unit operations are placed to dampen fluctuations in flow and pressure and also might be used as intermedia storage when a unit operation stops working. Fluctuations flow rate and concentrations are inherent when a conventional batch manufacturing process is rendered into a continuous. Then preferably perfusion culture is connected to a counter current chromatography. Constants harvest conditions in perfusion culture could be only expected if be fully growth arrested. This is not the case and therefore the excess of cells must be removed from the bioreactor by a so-called bleeding leading to a change in harvest flow rate. The concentration is simultaneously changing. Therefore without proper control these fluctuations would propagate through the entire process and eventually amplify and lead to a derailed process. Model predictive control ensures either constant harvest flow or concentration (Pappenreiter et al., 2022).
Conventional feedback control is not sufficient, because it is designed for rapid changes in process conditions, but in perfusion cell culture conditions change slowly (Zhao et al., 2015) . Then harvest is sent to a surge thank and then captured by a counter current chromatography. The loading flow rate is constant but elution after the columns is saturated leads to a periodically change of mass flow and in order to warrant a constant contact time in the virus inactivation the elution fractions are pooled and then very often subjected to a batch virus inactivation, although many continuous virus inactivation methods have been established. By following such a process design the benefits of continuous biomanufacturing are reduced in part automation and on-line control and real-time release become very difficult to implement. Surge tanks have a deteriorating effect RTD and wide RTD makes a process slow and batch definition difficult(Lali et al., 2022). In an ideal process the mass flow is never interrupted, and surge tanks are avoided. The perfusion bioreactor produces a constant harvest flow and can be further processed by flocculation and precipitation and polished with flow through chromatography methods. After concentration adjustment by e.g., single pass tangential flow filtration a continuous virus filtration can be added as recently suggested (Figure 9).