Materials and
Methods
Lab-scale system setup for pressure
characterization
An experimental setup capable of operating TFF, rTFF, HPTFF and scTFF
was built to characterize the different filtration systems at lab-scale
(Figure 1A ). Pressure transmitters (PREPS-N-038, PendoTECH,
Princeton, USA) were installed in the retentate loop at the inlet
(\(\text{PT}_{R1}\)) and outlet of the filter (\(\text{PT}_{R2}\)).
Further pressure transmitters were positioned in the filtrate loop at
the inlet (\(\text{PT}_{F1}\)) and outlet (\(\text{PT}_{F2}\)) of the
module. A PES hollow fiber-module with a total module length of 70 cm,
an effective fiber length of 65 cm, an inner fiber diameter of 1 mm, a
pore size of 0.2 μm and a membrane surface area of 0.15
m2 was used (S06-P20U-10-N, Repligen, Waltham,
Massachusetts, USA). For the pressure tests, the hollow fiber module was
modified by drilling holes and gluing additional pressure transmitters
(\(\text{PT}_{A1-5}\)) into the filtrate side to monitor the filtrate
pressures along the filter length. Considering the total length of the
hollow fiber module, the additional pressure transmitters
(\(\text{PT}_{A1-5}\)) were place at 5.5 cm, 20 cm, 35 cm, 50 cm and
64.5 cm measured from the start of the module. Flow sensors
(LFSC-i10X-001, Levitronix GmbH, Zurich, Switzerland) were installed in
the retentate loop (\(\text{FS}_{R}\)) and in the filtrate loop
(\(\text{FS}_{F}\)). Three levitated centrifugal pumps (PuraLev i30SU,
Levitronix GmbH, Zurich, Switzerland) were integrated into the
experimental setup. Two pumps were integrated into the retentate loop,
of which one was directed towards the filter inlet (\(\text{CD}_{R1}\))
and the other towards the filter outlet (\(\text{CD}_{R2}\)). The third
centrifugal pump (\(\text{CD}_{F}\)) was inserted into the filtrate loop
directed towards the filter inlet side. A peristaltic pump
(\(\text{PP}_{H}\)) was inserted into the harvest stream. Data was
recorded by connecting 3 process control units (LCO-i100, Levitronix
GmbH, Zurich, Switzerland).
Large-scale system setup for pressure
characterization
For large-scale pressure characterization, a similar experimental setup
as described for lab-scale experiments was built to achieve TFF, HPTFF
and scTFF operation (Figure 7AB ). A PES hollow fiber-module
with a total module length of 78 cm, an effective fiber length of 68 cm,
an inner fiber diameter of 1 mm, a pore size of 0.2 μm and a membrane
surface of 7.15 m2 was used (X06-P20U-10-N, Repligen,
Waltham, Massachusetts, USA). Pressure transmitters (PREPS-N-1-1,
PendoTECH, Princeton, USA) were installed in the retentate loop
(\(\text{PT}_{R1}\) and \(\text{PT}_{R2}\)) and in the filtrate loop
(\(\text{PT}_{F1}\) and \(\text{PT}_{F2}\)). To measure pressures as
closely as possible to the inlet and outlet of the hollow fibers, holes
were drilled to place pressure transmitters (PREPS-N-038, PendoTECH,
Princeton, USA) into the adapter piece of the filter module
(\(\text{PT}_{RC1}\) and \(\text{PT}_{RC2}\)). Further pressure
transmitters (PREPS-N-038, PendoTECH, Princeton, USA) on the filtrate
side were attached along the filter length at 15.5 cm, 27.5 cm, 38.5 cm,
50 cm and 62.5 cm measured from the start of the module
(\(\text{PT}_{A1-5}\)). Additionally, pressure transmitters at the
same filter length but in the back were attached
(\(\text{PT}_{AB1-5}\)). Flow sensors (LFSC-i35X, Levitronix GmbH,
Zurich, Switzerland) were installed in the retentate (\(\text{FS}_{R}\))
and filtrate loops (\(\text{FS}_{F}\)). One levitated centrifugal pump
(PuraLev-2000SU, Levitronix GmbH, Zurich, Switzerland) was inserted into
the retentate loop (\(\text{CD}_{R1}\)) and a second levitated
centrifugal pump (PuraLev-600SU, Levitronix GmbH, Zurich, Switzerland)
was placed in the filtrate loop (\(\text{CD}_{F}\)). A peristaltic pump
(\(\text{PP}_{H}\)) was inserted into the harvest stream. Data recording
was achieved by coupling 6 process control units (5x LCO-i100 and 1x
LCO-600, Levitronix GmbH, Zurich, Switzerland).
Experimental procedure for pressure
characterization
Pressure characterization studies were performed with water as a medium.
To characterize the TFF system, only pump \(\text{CD}_{R1}\) was
operational. To realize the HPTFF and scTFF systems, pump\(\text{CD}_{R1}\) in the retentate loop and pump \(\text{CD}_{F}\) in
the filtrate loop were active simultaneously. For the rTFF systems, pump\(\text{CD}_{R1}\) was used for the first phase and pump\(\text{CD}_{R2}\) was used for the second phase with reversed
crossflow. For all lab-scale setups, crossflow was kept at 650 mL/min,
except for ramping experiments where the crossflow was ramped from 0 –
1500 mL/min. For crossflow ramping in HPTFF, a delta pressure PI control
(P-term: 2 rpm/Δmbar; I-term: 5 rpm/Δmbar*sec) was used with a setpoint
of 0 mbar, meaning that the pump speed of pump (\(\text{CD}_{F}\)) was
controlled such that pressure \(\text{PT}_{F1}\) matched\(\text{PT}_{R1}\). For scTFF characterization, the co-current filtrate
flow rate was set at 870 mL/min (scTFF phase 1) and 1890 mL/min (scTFF
phase 2) to achieve a delta pressure control of ± 10 mbar. To
characterize large-scale TFF operation, the crossflow was ramped from 0
– 45 L/min. For HPTFF pressure characterization, additional filtrate
flow was applied by a delta pressure PI control (P-term: 2 rpm/Δmbar;
I-term: 5 rpm/Δmbar*sec) such that \(\text{PT}_{F1}\) was kept 6 mbar
higher than \(\text{PT}_{R1}\). Filtrate flow ramping to assess scTFF
operation was achieved by maintaining a crossflow of 14.5 L/min and
ramping the co-current filtrate flow from 0 – 70 L/min. To compare
filtration conditions between filtration module scales, shear rates
provided by the manufacturers lookup tool were considered (Repligen,
Waltham, Massachusetts, USA).
Perfusion culture process
A proprietary CHO-K1 cell line producing a bispecific mAb was expanded
in an incubator (Multitron, Infors HT, Bottmingen, Switzerland) for 21
days using a proprietary chemically defined perfusion platform medium
and an on-demand proprietary feed (Merck KGaA, Corsier-sur-Vevey,
Switzerland). Perfusion bioreactors (Labfors 5 Cell, Infors HT,
Bottmingen, Switzerland) were inoculated at a seeding density of 0.6 x
106 viable cells/mL. Culture conditions were
maintained at 36.5 °C with a dissolved oxygen setpoint at 50% (VisiFerm
DO Arc, Hamilton, Bonaduz, Switzerland). The pH was controlled at 7.07 ±
0.17 (EasyFerm Plus Arc probe Hamilton, Bonaduz, Switzerland) by
sparging CO2 and using a 1.1 M
Na2CO3 solution. Bioreactors were
operated at 2 L working volume. After an initial growth phase, an online
capacitance probe (Incyte Arc, Hamilton, Bonaduz, Switzerland) was used
to keep the viable cell volume (VCV) constant at 12%. Perfusion started
on day 0 and was kept constant at 1.3 reactor volumes per day until the
end of the process.
Cell retention devices in perfusion cell
culture
Bioreactor harvests were gravimetrically controlled to maintain the
bioreactor weight constant using the same hollow fiber module as
describes in section 2.1. Cell retention devices were either operated as
TFF (Figure 1B ), rTFF (Figure 1C ), HPTFF or scTFF
systems (Figure 1D ). In all setups, the crossflow was generated
by levitated centrifugal pumps (PuraLev i30SU, Levitronix GmbH, Zurich,
Switzerland) to ensure comparability of sieving studies. Pump speeds in
the retentate loop were set to 3500 rpm, initially corresponding to a
crossflow velocity of 650 mL/min. For TFF, HPTFF and scTFF operations,
the retentate loop pump (\(\text{CD}_{R1}\)) was stopped every 3 minutes
for 3 seconds to release accumulated air bubbles within the centrifugal
pump head. For HPTFF operations, a delta pressure PI control (P-term: 2
rpm/Δmbar; I-term: 5 rpm/Δmbar*sec) between pressure \(\text{PT}_{R1}\)and pressure \(\text{PT}_{F1}\) was used with a setpoint of 0 mbar to
control the speed of the filtrate pump (\(\text{CD}_{F}\)). As such,
filtrate pressures were matched with retentate pressures along the
filter length. scTFF operation was achieved by delta pressure control of
-5 mbar during scTFF phase 1 and +5 mbar during scTFF phase 2 between
pressure \(\text{PT}_{R1}\) and pressure \(\text{PT}_{F1}\). rTFF
operation consisted of two phases. In the first phase, the first
retentate pump (\(\text{CD}_{R1}\)) was operational and the second
retentate pump (\(\text{CD}_{R2}\)) was stopped. During the second
phase, pump (\(\text{CD}_{R2}\)) was operational and the pump
(\(\text{CD}_{R1}\)) was stopped. Phase times were 20 sec.
Reference analytics
Cell density, viability, cell diameter, and pH were measured using a
BioProfile FLEX2 (Nova Biomedical, Waltham, USA). Bioreactors were
automatically sampled by the FLEX2 On-Line Autosampler (Nova Biomedical,
Waltham, USA) and samples were fractionated using a Teledyne Cetac
ASX-7200 (Teledyne CETAC Technologies, Omaha, Nebraska, USA). Viable
cell volume was calculated as follows (Metze et al., 2020):