Discussion
Applying co-current filtrate flow represents a promising tool to
alleviate product retention in mammalian perfusion processes by
achieving uniform TMP conditions along the filter length. This decreases
(or eliminates) the Starling recirculation flow, reducing the filter
load by using the entire membrane surface (Radoniqi et al., 2018). In
this study, pressure characterization experiments demonstrated that
HPTFF operation is possible for a wide range of perfusion relevant
crossflows using levitated centrifugal pumps that provide uniform
(non-pulsatile) flow (Figure 2B ). By matching the inlet
retentate pressure \(\text{PT}_{R1}\ \)and the inlet filtrate pressure\(\text{PT}_{F1}\) with a simple delta pressure control to 0 mbar, a
uniform TMP was achieved along the lab-scale filter (Figure
2C ). Interestingly, the outlet filtrate pressure \(\text{PT}_{F2}\)showed increasing discrepancy from the retentate outlet pressure
(\(\text{PT}_{R2}\)) with increasing co-current filtrate flow. This
discrepancy may come from increasing turbulence at the filtrate outlet,
although it had no effect on the filtrate pressure profile
(\(\text{PT}_{A1-5}\)). A reduced setup consisting of two pressure
sensors (\(\text{PT}_{R1}\) and \(\text{PT}_{F1}\)), a retentate
centrifugal pump (\(\text{CD}_{R1}\)) and a filtrate centrifugal pump
(\(\text{CD}_{F}\)) are therefore sufficient to operate the HPTFF system
(Figure 1D ).
Large-scale pressure characterization revealed that HPTFF can also be
achieved with manufacturing scale filters (Figure 7 ). With the
filtration module used in this study, the inlet filtrate pressure\(\text{PT}_{F1}\) had to be increased by 6 mbar compared to the inlet
retentate pressure \(\text{PT}_{R1}\) to match the filtrate pressures\(\text{PT}_{A1-5}\) with the retentate pressure drop. A pressure
decrease from \(\text{PT}_{R1}\) positioned in the inlet retentate
tubing compared to the pressure sensor located in the adapter piece
connecting tube was observed (Figure 7EF ). This offset might be
due to the change in tube diameter from the inlet tubing to the much
wider adapter piece connecting to the hollow fiber module. Further,
pressure \(\text{PT}_{F1}\) had to be controlled higher than expected to
achieve HPTFF. As already observed in lab-scale, the outlet pressure
sensor in the filtrate loop \(\text{PT}_{F2}\) was lower than
anticipated. These findings might be explained by a combination of a
relatively smaller filtrate inlet adapter diameter than in the lab-scale
and perturbation of the flow pattern at elevated co-current filtrate
flows in the large-scale module. Nevertheless, determination of the
offset by pressure characterization allowed us to achieve HPTFF
operation across the entire tested crossflow range from 0 - 45 L/min by
only measuring pressures \(\text{PT}_{R1}\) and \(\text{PT}_{F1}\).
In perfusion cell culture, a uniform TMP, as per definition in HPTFF, is
not necessarily the highest priority as in protein separations (van
Reis, 1993; van Reis et al., 1997). The main objective in perfusion
processes is to avoid filter clogging and reduce product retention.
Therefore, a membrane sweep from time to time in the form of a backflush
can be beneficial to remove some deposited material, but avoiding
intense backflushing as attributed to irreversible fouling (Weinberger
& Kulozik, 2022). A novel operating mode was designed in this study
named stepping co-current TFF (scTFF). scTFF can be operated with the
same hardware setup as described for the HPTFF (Figure 1D ). By
lowering and subsequently increasing the co-current filtrate flow rate
compared to HPTFF operation, a TMP gradient was achieved along the
filter length resulting in a Starling recirculation (Figure
4A ). The Starling recirculation changed direction upon switching from
scTFF phase 1 to phase 2, generating a backflush on the first half of
the filter and then on the second half of the filter similar to what
occurs in ATF or rTFF operation (Pappenreiter et al., 2023; Radoniqi et
al., 2018; Weinberger & Kulozik, 2022). However, in contrast to ATF and
rTFF, where the strength of the Starling recirculation is a function of
crossflow velocity and filter length, the strength of the Starling
recirculation in scTFF can be tuned independently of both crossflow
velocity and fiber length. For demonstration, a TMP of ± 10 mbar was
targeted (Figure 4C ), but any other TMP larger or smaller can
be achieved just by varying the co-current filtrate flow rates
(Figure 4B ). Furthermore, scTFF can either be operated by
switching between scTFF phase 1 and phase 2 (Figure 4D) , or by
operating at HPTFF conditions and integrating a membrane sweeping from
time to time by lowering or increasing the co-current filtrate flow
(Figure 4E ). The duration of each phase can thereby freely be
chosen, giving even more operational flexibility. scTFF operation was
demonstrated at lab-scale, and pressure characterization experiments
revealed applicability at manufacturing scale without changing the
system setup (SI Figure 2 ).
Perfusion cell culture runs revealed significantly reduced product
sieving below 60% for TFF operation (Figure 5F ). This agrees
with the literature, where similarly reduced product sieving was
reported (Karst et al., 2016; Pappenreiter et al., 2023; Wang et al.,
2017a). It is worth mentioning that the average filtrate fluxes in this
study were particularly low with 0.6 L/m2/h compared
to commonly reported filtrate fluxes of 2-3 L/m2/h
(Radoniqi et al., 2018; Romann et al., 2023).
Despite similar pressure drop and therefore comparable absolute Starling
recirculation flow of rTFF compared to TFF, rTFF showed significantly
improved product sieving above 90%, which is comparable to what has
been observed in ATF systems (Pappenreiter et al., 2023). This confirms
that with an identical pump system, rTFF clearly outperformed TFF.
However, this study does not allow us to distinguish between the
beneficial contributions of backflushing at both the inlet and outlet,
utilization of the entire membrane surface, and / or relaxation of the
fouling deposit when the crossflow direction changes. The rTFF_2 run
with higher amount of cell culture debris showed lower product sieving
than rTFF_1 (Figure 5D ), highlighting that rTFF is still prone
to product retention which can likely be attributed to pronounced
fouling at the inlet or exit of the hollow fiber modules at elevated
debris levels (Sundar et al., 2023).
HPTFF operation entirely removing Starling recirculation due to a
uniform TMP along the filtration module showed similar or even higher
product sieving than rTFF operation. The HPTFF operation was interrupted
every 3 minutes for 3 seconds by stopping the crossflow to release
potentially trapped gas bubbles from the centrifugal pump head, which
might even have had a beneficial impact on product sieving. The slightly
delayed PI-response controlling the co-current filtrate flow resulted in
a quick sweep of the membrane, initially backflushing the membrane on
the first filter half, followed by backflushing of the second filter
half upon crossflow re-activation (Figure 6 ). Whereas bubble
trapping in the centrifugal pump head plays a minor role at larger
scales, intentional pump stopping from time to time to generate a
membrane sweep in HPTFF operation might still be an attractive option
(SI Figure 2D ). It must be mentioned that a similar effect can
be achieved by shortly increasing the crossflow by maintaining the
co-current filtrate flow PI control active.
Similar but more controlled sweeping of the membrane was alternatively
achieved by increasing or lowering the co-current filtrate flow at
constant crossflow (Figure 4A ). Intensity and location of the
backflush can be adjusted by changing the magnitude of the co-current
filtrate flow (Figure 4B ), offering a wide range of
possibilities not available in ATF or rTFF operation. scTFF allows the
Starling recirculation flow to be adjusted independently of filtration
module specifications or crossflow velocities without changing the
hardware setup. This novel approach enables further research to evaluate
the benefits of membrane sweeping in a controlled but flexible manner to
define best operating conditions depending on process requirements.
A critical aspect of unidirectional crossflow systems remains filter
inlet blocking (Weinberger & Kulozik, 2021a; Zydney, 2016). Cell clumps
or aggregates getting into the cell recirculation loop can be trapped at
the filter inlet blocking entire hollow fibers. In two unidirectional
crossflow runs (TFF_1 and HPTFF_2) filter inlet blocking led to
premature run termination. When working with cell lines prone to
aggregation, rTFF should be the chosen cell retention operation mode to
prevent inlet blocking by crossflow reversal. When aggregation is not an
issue and unidirectional crossflow represents no risk to premature run
termination, HPTFF or scTFF clearly outperform conventional TFF
operation. Further, HPTFF and scTFF offer greater flexibility compared
to ATF or rTFF systems by alleviating previously described restrictions
on filter characteristics and operation parameters:
- Crossflow velocity: No restriction to low crossflows as strategy to
avoid extensive Starling recirculation.
- Filtration module length: Enabling longer filtration modules due to
TMP control and thereby reducing system complexity with multiple
parallel modules.
- Inner fiber diameter: No need for increased inner fiber diameters to
reduce pressure gradient at the cost of membrane surface area or
greater hold-up volume.
- Pore size: Possibility to utilize larger pores sizes without
increasing Starling recirculation caused by lower membrane resistance.