Results

Pressure characterization of TFF, rTFF and HPTFF

Measurement of pressure gradients within the hollow fiber and on the filtrate side along the filter length for the TFF, rTFF and HPTFF system was performed with a special characterization setup enabling the operation of all three systems (Figure 1A ). This setup not only allowed us to measure the retentate loop and filtrate pressures at the inlet of the filter module (\(\text{PT}_{R1}\) and \(\text{PT}_{F1}\)) and at the outlet of the filter module (\(\text{PT}_{R2}\) and\(\text{PT}_{F2}\)), but enabled us to get additional measurements of the filtrate pressure along the filter module length (\(\text{PT}_{A1-5}\)). A crossflow ramping from 0 – 1500 mL/min to simulate the TFF system or one of the rTFF phases demonstrated that the axial pressure drop within the retentate loop increased with increasing crossflow as seen in the diverging retentate inlet pressure\(\text{PT}_{R1}\) and retentate outlet pressure \(\text{PT}_{R2}\)(Figure 2A ). All remaining pressure sensors on the filtrate side, irrespective of the crossflow, indicated the average pressure of\(\text{PT}_{R1}\) and \(\text{PT}_{R2}\). Aligning the pressures at a crossflow of 650 mL/min according to their position revealed positive TMP at the filter inlet and negative TMP at the filter outlet. The TMP was zero in the middle of the filtration module (Figure 2C ). A similar crossflow ramping was performed to characterize pressures for the HPTFF system with activated delta pressure control to match the filtrate inlet pressure \(\text{PT}_{F1}\) with the retentate inlet pressure \(\text{PT}_{R1}\) (Figure 2B ). In contrast to the TFF and rTFF, filtrate pressures along the filter were not identical anymore but matched the retentate pressure gradient along the entire filter length (Figure 2D ). The only filtrate pressure sensor with a discrepancy to the respective retentate pressure was \(\text{PT}_{F2}\). This discrepancy was negligible for crossflows below 400 mL/min but increased slightly with larger crossflow (Figure 2B ). Required co-current filtrate flows for varying crossflows using the lab-scale filter are provided in the supporting information (SI Figure 1A ).
Time resolved pressure recordings for the operation of a TFF and rTFF system at a crossflow of 650 mL/min are provided in Figure 2E . TFF is represented by only considering the forward crossflow phase. The rTFF is described by adding a reverse crossflow phase, and thereby alternating the crossflow. The only changing pressures upon crossflow reversal were the retentate pressures, with \(\text{PT}_{R1}\) taking the previous value of \(\text{PT}_{R2}\) and vice-versa. HPTFF operation at 650 mL/min crossflow was achieved upon delta pressure control activation with a co-current filtrate flow of approximately 1400 mL/min (Figure 2F ). Immediately, filtrate pressures align with the retentate pressure gradient and are stably maintained at the target values.
A schematic representation of the pressure characterization experiments summarizes the findings for the TFF system (Figure 3A ), rTFF system (Figure 3B ) and for the HPTFF system (Figure 3C ). The schematic pressure plots demonstrate the TMP differences along the fiber length for the TFF and the rTFF system. A zoomed view into a hollow fiber at the beginning, in the middle and at the end of the filter module further highlights the Starling recirculation indicated by arrows. Compared to the TFF and rTFF system, the filtrate pressures in the HPTFF system are well aligned with the retentate pressure thereby generating a uniform TMP of only slightly above zero along the length of the filtration module. Small arrows from retentate to filtrate indicate that the entire filtration area is utilized for filtration by avoiding Starling recirculation.

Pressure characterization of scTFF

Whereas HPTFF operation focused on matching filtrate pressures with the retentate pressure gradient and thereby removing Starling recirculation completely, we also examined a novel operating mode for unidirectional TFF defined as stepping co-current TFF (scTFF). The scTFF consists of two phases, a first phase with lower co-current filtrate flow than required for HPTFF, and a second phase with higher co-current filtrate flow, resulting in a step profile for the co-current filtrate flow (Figure 4A ). To demonstrate the impact of co-current filtrate flow on the pressure profiles, a co-current filtrate flow ramping was performed by fixing the crossflow to 650 mL/min (Figure 4B ). At 0 mL/min co-current filtrate flow, the system basically corresponded to a standard TFF operation. With increasing co-current filtrate flow, the pressure aligned more and more to the retentate pressure gradient and matched it at about 1400 mL/min, corresponding to the situation in HPTFF operation. Further increasing the co-current filtrate flow led to higher filtrate pressures in the first half of the filter and lower filtrate pressures in the second half of the filter compared to the retentate pressure gradient. The filtrate pressure at the outlet\(\text{PT}_{F2}\) was not plotted as similar discrepancies to the retentate pressure gradient as seen in Figure 2B were observed. Selecting a co-current filtrate flow of 870 mL/min for phase 1 (blue vertical dashed line) and 1890 mL/min for phase 2 (red vertical dashed line) of the scTFF operation, a delta pressure between\(\text{PT}_{R1}\) and \(\text{PT}_{F1}\) of -10 mbar and +10 mbar, respectively, was achieved. Pressures recorded for the two phases of scTFF were then plotted according to their position along the filter (Figure 4C ). The black line represents the retentate pressure gradient, the blue dashed line represents the pressure drop on the filtrate side for scTFF phase 1 and the red line represents the pressure drop on the filtrate side for scTFF phase 2. A common intersection of all three lines was located in the middle of the filter length, meaning the absolute TMP is zero in the middle of the filter and gets larger the closer to one of two filter ends.
By switching between scTFF phase 1 and scTFF phase 2 with defined phase times, a scTFF system with unidirectional crossflow but reversing Starling recirculation was obtained (Figure 4C ). Red areas represent the flux of filtrate back into the retentate due to higher filtrate pressures compared to the retentate pressures, whereas blue areas represent flux from retentate to filtrate due to higher retentate pressures compared to filtrate pressures. As such, filtrate pressure\(\text{PT}_{A1}\) positioned at 5.5 cm from the filter inlet was lower than the corresponding retentate pressure at 5.5 cm filter length (black dashed line) during scTFF phase 1 and got larger than the corresponding retentate pressure during scTFF phase 2. Similar, but reversed, behaviour was observed for pressure \(\text{PT}_{A5}\) positioned on the second half of the filter at 64.5 cm filter length. In this case,\(\text{PT}_{A5}\) was larger than the retentate pressure during scTFF phase 1 and smaller than the retentate pressure during scTFF phase 2. A combined HPTFF-scTFF operation is also possible by integrating a sweeping into the HPTFF operation. The sweeping was achieved by lowering the co-current filtrate flow (scTFF phase 1) and subsequently increasing the co-current filtrate flow (scTFF phase 2). After the sweep, the system was again operated at HPTFF conditions (Figure 4D ).

Characterization of performance in perfusion cell culture processes

Cell culture parameters and product retention were compared for TFF, rTFF, HPTFF and scTFF operation in steady-state perfusion processes. For all four cell retention setups (Figure 1B-D ), steady-state operation was achieved after approximately 5 days and culture viability was not impacted by the cell retention operating mode (Figure 5A ). Target process run time of 30 days was achieved for all runs except TFF_1 and HPTFF_2. These runs were terminated at day 19 (TFF_1) and day 21 (HPTFF_2) due to a sudden decrease in crossflow caused by inlet blocking of the fibers. Cell diameter increased slightly with runtime for all of the cell retention systems (Figure 5B ) and pH stayed within the defined range of 7.07 ± 0.17 for all runs (Figure 5C ). Cell debris increased for most runs until day 25, after which a slight decrease in cell debris was observed. In general, TFF and rTFF runs showed slightly higher debris levels compared to HPTFF and scTFF runs especially after day 13 (Figure 5D ). The harvest titer plot (Figure 5E ) and the product sieving plot (Figure 5F ) revealed significantly reduced product sieving of around 80% for the TFF operation after only a few first days of steady-state operation. Product sieving further decreased down to 60% or lower for TFF. Product sieving for rTFF stayed above 90% for the entire experiment for run rTFF_1 and remained above 80% for run rTFF_2. HPTFF operation resulted in similar or even higher product sieving with yields above 95% for the entire run.
Cell culture bioreactors must be oxygenated to support cell growth by sparging air or oxygen. Centrifugal pumps in unidirectional crossflow operations (TFF, HPTFF and scTFF) tend to accumulate gas bubbles coming into the cell recirculation loop. This problem was solved by stopping the pumps for 3 seconds every 3 minutes to release the air from the pump head. With activated delta pressure control during HPTFF operation controlling delta pressure to 0 mbar, stopping the crossflow for 3 seconds caused a sharp change in the pressure profile along the filter length (Figure 6A ). Due to some delay of the PI controlled co-current filtrate flow regulation, the filtrate pressure\(\text{PT}_{F1}\) was higher than the retentate pressure\(\ \text{PT}_{R1}\) immediately after crossflow stopping, which resulted in a negative delta pressure up to -14 mbar (Figure 6Bred area). After reactivation of the crossflow, the co-current filtrate flow was reduced and the PI control required some more time to establish HPTFF conditions. During that time interval, a positive delta pressure of up to 5 mbar was seen at the filter inlet (Figure 6B blue area). Taken together, stopping the crossflow during HPTFF operation resulted in a slight membrane sweeping. In rTFF operation, gas bubble trapping was alleviated by positioning the pumps such that they are pointing towards each other, with air removed from the pump head by alternating activation of the retentate pumps.

Large-scale filter pressure characterization

Large-scale experiments using TFF, HPTFF and scTFF operation confirmed results from the lab-scale experiments. Crossflow ramping from 0 – 45 L/min showed a continued increase in the pressure gradient along the filter length in TFF operation while the permeate pressures were independent of position (Figure 7B ). Considering similar crossflow conditions with 29 L/min corresponding to a shear rate of 1470 s-1 as applied during lab-scale perfusion cell culture runs resulted in a fiber inlet pressure of 71 mbar \(\text{PT}_{RC1}\), a fiber outlet pressure of 31 mbar (\(\text{PT}_{RC2}\)) and an average filtrate pressure (\(\text{PT}_{A1-5}\)) of 51 mbar (Figure 7D ). Filtrate pressures on the filtrate inlet and outlet (\(\text{PT}_{A1-5}\)) showed similar values as the filtrate pressure sensors on the backside of the filter module (\(\text{PT}_{AB1-5}\)).
HPTFF operation could be achieved by controlling pressure\(\text{PT}_{F1}\) so that it was 6 mbar above the pressure\(\text{PT}_{R1}\) for all evaluated crossflows up to 45 L/min (Figure 7D ). Filtrate pressures on the filtrate inlet and outlet (\(\text{PT}_{A1-5}\)) were aligned with the corresponding filtrate pressures on the backside (\(\text{PT}_{AB1-5}\)) (Figure 7F ). In contrast to TFF operation, filtrate pressures matched the pressure gradient of the retentate loop, with pressure sensors \(\text{PT}_{A1}\) and\(\text{PT}_{AB1}\) slightly lower than the corresponding retentate pressures. The filtrate outlet pressure \(\text{PT}_{F2}\ \)was significantly lower compared to the other pressure sensor readings. Co-current filtrate flows to achieve HPTFF at varying crossflows are provided in the supporting information section (SI Figure 1B).
scTFF operation to generate controlled Starling recirculation was further demonstrated with a large-scale filter module and the data are provided in the supporting information section (SI Figure 2 ). As such, a filtrate loop ramping at constant crossflow (SI Figure 2A ) and pressure distribution along the filter length for scTFF phase 1 (SI Figure 2B ) and for scTFF phase 2 (SI Figure 2C ) are provided. In addition to changing the filtrate flow, a crossflow stop whilst keeping the filtrate PI control active was able to achieve effective membrane sweeping (SI Figure 2D ).