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\begin{document}

\title{Co-current filtrate flow in TFF perfusion processes: Decoupling
transmembrane pressure from crossflow to improve product sieving}



\author[1]{Patrick Romann}%
\author[1]{Philip Giller}%
\author[2]{Antony Sibilia}%
\author[3]{Christoph Herwig}%
\author[4]{Negin Nejatishahidein}%
\author[5]{Arnaud Perilleux}%
\author[5]{Jonathan Souquet}%
\author[5]{Jean-Marc Bielser}%
\author[1]{Thomas K. Villiger}%
\affil[1]{Fachhochschule Nordwestschweiz FHNW - Campus Muttenz}%
\affil[2]{Levitronix GmbH}%
\affil[3]{Technische Universitat Wien}%
\affil[4]{The Pennsylvania State University Department of Chemical Engineering}%
\affil[5]{Merck Serono SA Aubonne}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Hollow fiber-based membrane filtration has emerged as the dominant
technology for cell retention in perfusion processes yet significant
challenges in alleviating filter fouling remain unsolved. In this work,
the benefits of co-current filtrate flow applied to a tangential flow
filtration (TFF) module to reduce or even completely remove Starling
recirculation caused by the axial pressure drop within the module was
studied by pressure characterization experiments and perfusion cell
culture runs. Additionally, a novel concept to achieve alternating
Starling flow within unidirectional TFF was investigated. Pressure
profiles demonstrated that precise flow control can be achieved with
both lab-scale and manufacturing scale filters. TFF systems with
co-current flow showed up to 40\% higher product sieving compared to
standard TFF. The decoupling of transmembrane pressure from crossflow
velocity and filter characteristics in co-current TFF alleviates common
challenges for hollow-fiber based systems such as limited crossflow
rates and relatively short filter module lengths, both of which are
currently used to avoid extensive pressure drop along the filtration
module. Therefore, co-current filtrate flow in unidirectional TFF
systems represents an interesting and scalable alternative to standard
TFF or alternating TFF operation with additional possibilities to
control Starling recirculation flow.%
\end{abstract}%



\sloppy


Co-current filtrate flow in TFF perfusion processes: Decoupling
transmembrane pressure from crossflow to improve product sieving Patrick
Romann\textsuperscript{1,2}, Philip Giller\textsuperscript{1}, Antony
Sibilia\textsuperscript{3}, Christoph Herwig\textsuperscript{2}, Andrew
L. Zydney\textsuperscript{4}, Arnaud Perilleux\textsuperscript{5},
Jonathan Souquet\textsuperscript{5}, Jean-Marc
Bielser\textsuperscript{5}, and Thomas K.
Villiger\textsuperscript{1*1}Institute for Pharma Technology, School of
Life Science, University of Applied Sciences and Arts Northwestern
Switzerland, Muttenz, Switzerland\textsuperscript{2}Research Division
Biochemical Engineering, Institute of Chemical Environmental and
Bioscience Engineering, Vienna University of Technology,
Austria\textsuperscript{3}Levitronix GmbH, Z\selectlanguage{ngerman}ürich,
Switzerland\textsuperscript{4}Department of Chemical Engineering, The
Pennsylvania State University, University Park, 16802 Pennsylvania,
USA\textsuperscript{5}Biotech Process Science, Merck Serono SA (an
affiliate of Merck KGaA, Darmstadt, Germany), Corsier-sur-Vevey,
Switzerland *To whom correspondence should be addressed: Email:
thomas.villiger@fhnw.ch

\emph{}

\emph{\textbf{Abstract:}} Hollow fiber-based membrane filtration has
emerged as the dominant technology for cell retention in perfusion
processes yet significant challenges in alleviating filter fouling
remain unsolved. In this work, the benefits of co-current filtrate flow
applied to a tangential flow filtration (TFF) module to reduce or even
completely remove Starling recirculation caused by the axial pressure
drop within the module was studied by pressure characterization
experiments and perfusion cell culture runs. Additionally, a novel
concept to achieve alternating Starling flow within unidirectional TFF
was investigated. Pressure profiles demonstrated that precise flow
control can be achieved with both lab-scale and manufacturing scale
filters. TFF systems with co-current flow showed up to 40\% higher
product sieving compared to standard TFF. The decoupling of
transmembrane pressure from crossflow velocity and filter
characteristics in co-current TFF alleviates common challenges for
hollow-fiber based systems such as limited crossflow rates and
relatively short filter module lengths, both of which are currently used
to avoid extensive pressure drop along the filtration module. Therefore,
co-current filtrate flow in unidirectional TFF systems represents an
interesting and scalable alternative to standard TFF or alternating TFF
operation with additional possibilities to control Starling
recirculation flow.\textbf{Keywords:} Perfusion cell culture, tangential
flow filtration (TFF), co-current filtrate flow, Starling recirculation,
product sieving

\section*{Introduction}

{\label{introduction}}

Hollow fiber-based tangential flow filtration has emerged as one of the
most preferred cell retention technologies for mammalian perfusion
processes with applications in main stage perfusion bioreactors and also
in N-1 bioreactors improving existing fed-batch production units
(Bielser et al., 2018; Coffman, Brower, Connell\selectlanguage{english}-Crowley, et al., 2021;
Wolf et al., 2020). Despite significant advancements in pump technology,
such as low-shear diaphragm pumps or levitated centrifugal pumps
(Blaschczok et al., 2013; Clincke et al., 2013; Kelly et al., 2014; Wang
et al., 2017a), to improve culture viability and thereby reduce the load
of fouling-provoking particles on the filter membrane, filter clogging
and product retention remain major challenges on the way to robust
manufacturing processes.

Tangential flow filtration (TFF) in unidirectional crossflow mode
(frequently driven by a levitated centrifugal pump) and alternating
tangential flow (ATF, driven by a diaphragm pump) are the most commonly
reported systems in perfusion processes (Fisher et al., 2019; MacDonald
et al., 2022; Matanguihan \& Wu, 2022). Most studies revealed that ATF
showed superior product sieving compared to TFF at lab-scale and
suggested ATF as a more suitable technology for long-term perfusion
operation (Clincke et al., 2013; Karst et al., 2016; Wang et al.,
2017a). However, ATF systems driven by diaphragm pumps were associated
with operational instability at manufacturing scale (Coffman, Brower,
Connell-Crowley, et al., 2021; Pavlik, 2017, 2019; Shevitz, 2018).
Furthermore, multiple parallel ATF system were required to operate
2000~L perfusion bioreactors, requiring considerably more floor space
compared to similar TFF systems (Coffman, Brower, Connell\selectlanguage{english}-Crowley, et
al., 2021; Romann et al., 2023). Therefore, TFF systems were claimed to
be the preferred choice within the industry at large scale due to
smaller facility footprint and higher robustness, whereas ATF systems
showed improved product sieving and reduced development time for
pilot-scale operations (Coffman, Brower, Connell-Crowley, et al., 2021).

In TFF, concentration polarization and fouling can both affect product
retention (Belfort et al., 1994; Chew et al., 2020; Field, 2010; Taddei
et al., 1990; van Reis \& Zydney, 2007). Compared to industrial TFF
applications that have short operating times and high filtrate flux
(Redkar \& Davis, 1993; Tanaka et al., 1997; Weinberger \& Kulozik,
2021b), TFF systems used as cell retention devices in perfusion
processes must be operational for as much as several months (without
cleaning) and are operated at comparably very low filtrate fluxes of
around 2 L/m\textsuperscript{2}/h (Radoniqi et al., 2018). Due to the
low filtrate fluxes and high axial pressure drops, a reverse flow of
filtrate back into the fiber lumen occurs at the filter exit. This
so-called Starling recirculation (Starling, 1896) was modelled and shown
to be significantly larger than the actual harvest rate during typical
perfusion processes (Radoniqi et al., 2018). As a consequence, only
slightly more than 50\% of the actual hollow fiber membrane surface area
is used for filtration (Radoniqi et al., 2018).

More recently, alternating TFF systems have been described either with
one levitated centrifugal pump and valves to switch crossflow direction
(Weinberger \& Kulozik, 2021a), or with two alternating, inversely
positioned centrifugal pumps in the retentate loop called reverse TFF
(rTFF) (Pappenreiter et al., 2023; Weinberger \& Kulozik, 2022). Both
setups showed reduced product retention compared to unidirectional TFF
systems. While each individual phase of alternating crossflow filtration
(ATF, rTFF or alternating crossflow by valve switching) can be compared
to the situation in a TFF system, the distinguishing factor lies in the
alternating direction of the crossflow and therefore the change in the
location of the filter inlet. When working with cell lines prone to
aggregation, switching the filter inlet can prevent fiber blocking
(Weinberger \& Kulozik, 2021a; Zydney, 2016). Improved product sieving
with alternating crossflow systems compared to TFF was further
attributed to the short period of zero net flow between phases resulting
in a very low TMP across the entire filter length, possibly leading to
deposit layer relaxation (Weinberger \& Kulozik, 2021a, 2021b). It was
also suggested that the Starling recirculation, which switches between
the two ends of the hollow fiber, could remove deposited material and
thereby minimize fouling (Karst et al., 2016; Radoniqi et al., 2018).
Additionally, alternating crossflow filtration makes use of the entire
filter length, harnessing the full membrane surface of the module
(Radoniqi et al., 2018). The increased performance of alternating TFF is
likely due to a combination of these factors.

Although there are several advantages associated with alternating
crossflow filtration, it is important to note that the backflush of
filtrate at the filter exit must be counterbalanced by an increased
filtrate flux near the filter inlet to maintain the same overall level
of filtration. This causes an increase in the drag forces that push
particles into the membrane (Ripperger \& Altmann, 2002), which can lead
to a denser deposit and greater particle penetration into the membrane,
both detectable as an increase of irreversible fouling resistance
(Weinberger \& Kulozik, 2022). Sundar et al. (2023) demonstrated that
the greatest fouling in ATF systems occurs at the ends of the hollow
fiber module, i.e., where the filtrate flux is greatest. This might
explain studies observing significant product sieving losses despite
using properly sized ATF modules (Kim et al., 2016; Wang et al., 2017b).

These phenomena provide notable constraints in hollow fiber designs and
determining optimal operating conditions for perfusion systems.
Crossflow velocities, for example, must be kept low to decrease the
pressure drop along the filter length and thereby reduce the Starling
recirculation. However, reducing crossflow increases the residence time
of the cells within the recirculation loop, risking oxygen depletion
(Walther et al., 2019). In addition, low crossflow leads to greater
concentration polarization, i.e., greater accumulation of cells at the
membrane surface. The increasing axial pressure drop with increasing
length of the hollow fiber modules favors the use of relatively short
filtration modules, requiring multiple filters in parallel to meet the
needs for larger filtration area. Increasing the inner diameter of the
hollow fibers would also reduce the pressure drop along the fiber
length, but at a cost of much greater hold-up volume within the module.

Although all these strategies to improve filtration performance try to
reduce the impact of the pressure drop and Starling flow, none of them
solve the fundamental problem that the local TMP is coupled to the
magnitude of the crossflow (which determines the axial pressure drop)
and the filter characteristics. In order to achieve a nearly uniform TMP
throughout the module, a similar pressure drop must be generated on the
filtrate side of the module as on the retentate side. This concept for
the biopharmaceutical industry was originally called High Performance
TFF (HPTFF) and was successfully demonstrated to control concentration
polarization along the filter length to enable high resolution
protein-protein separations in downstream operations (van Reis, 1993;
van Reis et al., 1997) . It has also improved purification of viral
vectors using ultrafiltration (Grzenia et al., 2008). The concept is
further known in the dairy industry for microfiltration (Merin \&
Daufin, 1990; Sandblom, 1978; Vadi \& Rizvi, 2001). The ability to
control the filtrate flux and Starling flow independently of the
crossflow and length of the filtration module offers a promising tool to
overcome challenges in current TFF and ATF systems in perfusion
processes. To the authors knowledge, HPTFF to alleviate product
retention in perfusion processes has not been evaluated in the
literature.

The aim of this study was to develop a co-current filtration flow system
for perfusion processes based on single-use low-shear centrifugal pumps
in combination with pressure sensors with the ultimate goal to reduce
product retention. This so-called HPTFF system was characterized for a
wide range of operating conditions by measuring the TMP along the length
of the filter module. Steady-state perfusion cell culture runs
demonstrated superior performance during HPTFF compared to standard TFF.
Further, a new concept of stepping co-current TFF (scTFF) was
introduced, allowing us to generate alternating Starling recirculation
within a unidirectional TFF system. Subsequently, scalability of HPTFF
and scTFF were successfully evaluated by recording pressure profiles
along a modified large-scale hollow fiber module.

\section*{Materials and Methods}

{\label{materials-and-methods}}

\subsection*{Lab-scale system setup for pressure
characterization}

{\label{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
(\textbf{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 \selectlanguage{greek}μ\selectlanguage{english}m and a membrane surface area of 0.15
m\textsuperscript{2} 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).

\subsection*{Large-scale system setup for pressure
characterization}

{\label{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 (\textbf{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 \selectlanguage{greek}μ\selectlanguage{english}m and a membrane
surface of 7.15 m\textsuperscript{2} 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).

\subsection*{Experimental procedure for pressure
characterization}

{\label{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/\selectlanguage{greek}Δ\selectlanguage{english}mbar; I-term: 5 rpm/\selectlanguage{greek}Δ\selectlanguage{english}mbar*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 \selectlanguage{ngerman}± 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/\selectlanguage{greek}Δ\selectlanguage{english}mbar;
I-term: 5 rpm/\selectlanguage{greek}Δ\selectlanguage{english}mbar*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).

\subsection*{Perfusion culture process}

{\label{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
10\textsuperscript{6} viable cells/mL. Culture conditions were
maintained at 36.5 \selectlanguage{ngerman}°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 CO\textsubscript{2} and using a 1.1~M
Na\textsubscript{2}CO\textsubscript{3} 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.

\subsection*{Cell retention devices in perfusion cell
culture}

{\label{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 (\textbf{Figure 1B} ), rTFF (\textbf{Figure 1C} ), HPTFF or scTFF
systems (\textbf{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/\selectlanguage{greek}Δ\selectlanguage{english}mbar; I-term: 5 rpm/\selectlanguage{greek}Δ\selectlanguage{english}mbar*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.

\subsection*{Reference analytics}

{\label{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):\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\(VCV=\frac{4}{3}\bullet\pi\bullet\left(\frac{D}{2}\right)^{3}\bullet\text{VCD}\bullet 100\) & (3)\tabularnewline
\bottomrule
\end{longtable}

where D is the average cell diameter and VCD is the viable cell density,
assuming a spherical shape of the cells. Bioreactor titer samples were
prepared by centrifugation (3200 g for 10 min) and 0.22 \selectlanguage{greek}μ\selectlanguage{english}m filtered.
Harvest titer samples were directly analyzed without further sample
preparation. Process titers were determined using a protein A affinity
high performance liquid chromatography device (PA-HPLC, Waters, Milford,
Massachusetts, USA). Cell debris was measured using an optical density
(OD) measurement (Spectronic Genesys 10 Bio, Thermo Electron
Corporation, Waltham, Massachusetts, USA). Therefore, bioreactor samples
were centrifuged (3200 g for 10 min) and measured at 600 nm against a
0.22 \selectlanguage{greek}μ\selectlanguage{english}m filtered reference sample.

\section*{Results}

{\label{results}}

\subsection*{Pressure characterization of TFF, rTFF and
HPTFF}

{\label{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 (\textbf{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
\(\text{PT}_{R2}\)(\textbf{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
(\textbf{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 \(\text{PT}_{R1}\) (\textbf{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 (\textbf{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
(\textbf{Figure 2B} ). Required co-current filtrate flows for varying
crossflows using the lab-scale filter are provided in the supporting
information (\textbf{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 \textbf{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 (\textbf{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 (\textbf{Figure 3A} ), rTFF
system (\textbf{Figure 3B} ) and for the HPTFF system (\textbf{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.

\subsection*{Pressure characterization of
scTFF}

{\label{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
(\textbf{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 (\textbf{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 \textbf{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
(\textbf{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 (\textbf{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
(\textbf{Figure 4D} ).

\subsection*{Characterization of performance in perfusion cell culture
processes}

{\label{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 (\textbf{Figure 1B-D} ), steady-state
operation was achieved after approximately 5 days and culture viability
was not impacted by the cell retention operating mode (\textbf{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 (\textbf{Figure 5B} )
and pH stayed within the defined range of 7.07 \selectlanguage{ngerman}± 0.17 for all runs
(\textbf{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 (\textbf{Figure 5D} ). The
harvest titer plot (\textbf{Figure 5E} ) and the product sieving plot
(\textbf{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 (\textbf{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 (\textbf{Figure
6B}red 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 (\textbf{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.

\subsection*{Large-scale filter pressure
characterization}

{\label{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 (\textbf{Figure 7B} ). Considering similar
crossflow conditions with 29 L/min corresponding to a shear rate of 1470
s\textsuperscript{-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 (\textbf{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
(\textbf{Figure 7D} ). Filtrate pressures on the filtrate inlet and
outlet (\(\text{PT}_{A1-5}\)) were aligned with the corresponding
filtrate pressures on the backside (\(\text{PT}_{AB1-5}\)) (\textbf{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
(\textbf{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 (\textbf{SI Figure 2} ).
As such, a filtrate loop ramping at constant crossflow (\textbf{SI
Figure 2A} ) and pressure distribution along the filter length for scTFF
phase 1 (\textbf{SI Figure 2B} ) and for scTFF phase 2 (\textbf{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 (\textbf{SI Figure 2D} ).

\section*{Discussion}

{\label{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 (\textbf{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
(\textbf{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 (\textbf{Figure 1D} ).

Large-scale pressure characterization revealed that HPTFF can also be
achieved with manufacturing scale filters (\textbf{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 (\textbf{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 (\textbf{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 (\textbf{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 (\textbf{Figure 4C} ), but any other TMP larger or smaller can
be achieved just by varying the co-current filtrate flow rates
(\textbf{Figure 4B} ). Furthermore, scTFF can either be operated by
switching between scTFF phase 1 and phase 2 (\textbf{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
(\textbf{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 (\textbf{SI Figure 2} ).

Perfusion cell culture runs revealed significantly reduced product
sieving below 60\% for TFF operation (\textbf{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/m\textsuperscript{2}/h compared
to commonly reported filtrate fluxes of 2-3 L/m\textsuperscript{2}/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 (\textbf{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 (\textbf{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
(\textbf{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 (\textbf{Figure 4A} ). Intensity and location of the
backflush can be adjusted by changing the magnitude of the co-current
filtrate flow (\textbf{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:

\begin{enumerate}
\tightlist
\item
  Crossflow velocity: No restriction to low crossflows as strategy to
  avoid extensive Starling recirculation.
\item
  Filtration module length: Enabling longer filtration modules due to
  TMP control and thereby reducing system complexity with multiple
  parallel modules.
\item
  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.
\item
  Pore size: Possibility to utilize larger pores sizes without
  increasing Starling recirculation caused by lower membrane resistance.
\end{enumerate}

\section*{Conclusion}

{\label{conclusion}}

This study evaluated the impact of co-current filtrate flow on product
retention during steady-state perfusion processes using hollow fiber
modules as cell retention devices. Whereas Starling recirculation in TFF
and alternating crossflow TFF is dependent on crossflow velocity and
filter module characteristics, co-current filtrate flow enabled
independent control of Starling recirculation. Pressure characterization
studies performed by inserting additional pressure sensors along the
filter module length revealed detailed insights into the filtrate
pressure gradient and confirmed the theoretical concept of altering TMP
by co-current filtrate flow. Further, control of Starling flow was not
only demonstrated at lab-scale, but also successfully applied to a
manufacturing scale filtration module. The benefits of HPTFF operation
or precisely controlling the direction and intensity of Starling
recirculation in scTFF operation was further demonstrated in
steady-state perfusion cell culture processes which showed much higher
product sieving compared to standard TFF operation. Starling flow
control enabled by co-current filtrate flow operation represents an
effective tool not only to study filter fouling, but also to reduce
product retention in steady-perfusion cell culture processes as well as
other operations such as dynamic perfusion or N-1 perfusion.

List of Abbreviations

TFF Tangential Flow Filtration

ATF Alternating Tangential Flow Filtration

TMP Transmembrane Pressure

rTFF Reverse Tangential Flow Filtration

HPTFF High Performance Tangential Flow Filtration

scTFF Stepping Co-current Tangential Flow Filtration

PT\textsubscript{R1-2} Pressure Transmitter in retentate loop

PT\textsubscript{F1-2} Pressure Transmitter in filtrate loop

PT\textsubscript{A1-5} Additional Pressure Transmitter on filtrate side
of hollow fiber filter

PT\textsubscript{AB1-5} Additional Pressure Transmitter on backside of
filtrate side of hollow fiber filter

FS\textsubscript{R} Flow Sensor in retentate loop

FS\textsubscript{F} Flow Sensor in filtrate loop

CD\textsubscript{R1-2} Centrifugal Discharge Pump in retentate loop

CD\textsubscript{F} Centrifugal Discharge Pump in filtrate loop

PP\textsubscript{H} Peristaltic Pump in harvest line

VCV Viable Cell Volume

VCD Viable Cell Density

Potential conflict of interest

Antony Sibilia is an employee of Levitronix GmbH, a company which
produces and markets centrifugal pumps and flow sensors used in the
present study. He was not involved in the interpretation of data or
statistical analysis. All other authors declare that they have no known
competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.\emph{}

Author Contributions:

\textbf{Patrick Romann:} Conceptualization, formal analysis,
investigation, methodology, visualization, writing -- original draft

\textbf{Philip Giller:} Data curation, formal analysis, investigation,
methodology

\textbf{Christoph Herwig:} Conceptualization, methodology, supervision,
writing -- review \& editing

\textbf{Andrew L. Zydney:} Conceptualization, methodology, supervision,
writing -- review \& editing

\textbf{Arnaud Perilleux:} resources, supervision, writing -- review \&
editing

\textbf{Jonathan Souquet:} resources, supervision, writing -- review \&
editing

\textbf{Jean-Marc Bielser:} Conceptualization, investigation, project
administration, resources, supervision, visualization, writing -- review
\& editing

\textbf{Thomas K. Villiger:} Conceptualization, investigation, project
administration, resources, supervision, visualization, writing -- review
\& editing

Acknowledgements

The authors would like to thank Pavel Dagorov and his workshop team
(FHNW) for hollow fiber filter modification and Sebastian Schneider and
Silvia Pavone (FHNW) for supporting experimental work on perfusion
processes. The authors also thank the entire Levitronix team for
support, especially Knut Kuss for valuable input. Dominik Schieman, Paul
Niepold and David Garcia (Novartis) kindly sponsored the manufacturing
scale filter module. Moreover, the authors would like to acknowledge
specifically the Bioprocess Sciences (BPS) team of Merck Serono SA (an
affiliate of Merck KGaA) for material and analytical support and for
valuable discussions and support throughout the project, especially
Alexandre Châtelin and the analytical team.

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\begin{longtable}[]{@{}l@{}}
\toprule
\textbf{Figure 1} Detailed schematic representation of the experimental
setup for the pressure characterization in water (A). Pressure
transmitters (PT), flow sensors (FS), centrifugal pumps (CD) and
peristaltic pumps (PP) are specified with subscripted letters according
to their position (R: Retentate; F: Filtrate; A: Additional sensors on
filtrate side; H: Harvest). Minimal required setups for TFF (B), rTFF
(C) and HPTFF or scTFF (D) operation used for cell culture runs are
further shown.\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
\textbf{Figure 2} Pressure characterization results for TFF, rTFF and
HPTFF operation. Crossflow ramping in TFF and rTFF operation (A) and
crossflow ramping with delta pressure control in HPTFF operation (B).
Dashed lines represent the standard operating region of 1470
s\textsuperscript{-1} shear resulting in approximately 650 mL/min
crossflow. Pressure measurements according to their position along the
filter length are provided for TFF and the forward crossflow phase of
rTFF (C), and for HPTFF (D), grey areas represent absolute fiber length.
Pressure distributions at 650 mL/min crossflow versus time are shown for
TFF and rTFF (E), where the forward crossflow phase corresponds to TFF
operation, and rTFF is defined by alternation between forward and
reverse crossflow. HPTFF pressure distribution versus time upon
co-current filtrate flow activation was further measured at 650 mL/min
crossflow (F).\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
\textbf{Figure 3} Schematic representation for TFF (A), rTFF (B) and
HPTFF (C) systems with centrifugal pumps based on pressure
characterization. Pressure curves along the filter length for each
system are given and arrows indicate filtrate flux, longer arrows
represent larger fluxes. Additionally, a zoom into a fiber at the
beginning, middle and at the end of the filter is provided and colored
from red (high pressure) to blue (low pressure). As rTFF consist of two
phases, the situation for forward crossflow and reverse crossflow are
depicted.\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
\textbf{Figure 4} Concept of stepping co-current TFF (scTFF) by
schematic representation of pressure gradient along filter length (A).
Co-current flow ramping at 650 mL/min crossflow (B). Vertical dashed
lines represent operating conditions for HPTFF (black), scTFF phase 1
with -10 mbar pressure difference (blue) and scTFF phase 2 with +10 mbar
pressure difference (red) on filter inlet. Pressure measurements
according to their position along the filter length are provided for all
three dashed lines (C), grey areas represent absolute fiber length.
Operation of scTFF with two alternating phases (\selectlanguage{ngerman}±10 mbar pressure
difference) is demonstrated by time resolved pressure distribution plots
(D). Alternative operating mode of HPTFF with alternating membrane
sweeps by scPTFF phases of ± 10 mbar in between HPTFF phases (E). Red
areas represent flux of filtrate back into the retentate, blue areas
represent flux from retentate to filtrate.\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
\textbf{Figure 5} Perfusion cell culture runs with TFF (black), rTTF
(blue), HPTFF or scTFF (red) as cell retention devices. VCV and
viability (A), cell diameter (B), pH (C), cell culture debris (D),
harvest titer (E) and product sieving (F).\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
\textbf{Figure 6} Membrane sweep in HPTFF operation upon crossflow stop.
Inlet pressure of the retentate (\(\text{PT}_{R1}\)) and inlet pressure
on filtrate side (\(\text{PT}_{F1}\)) were recorded upon a 3 sec stop of
the levitated centrifugal pump (\(\text{CD}_{R1}\)) to release air
bubbles (A). The delta pressure (\(\text{PT}_{R1}-\text{PT}_{F1}\)) was calculated and
the blue area represents a negative delta pressure during which a
backflush is happening in the first half of the filter, whereas the red
area represents a positive delta pressure resulting in a backflush at
the second half of the filter (B).\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
\textbf{Figure 7} Large-scale pressure characterization results.
Schematic representation of the experimental setup for the large-scale
pressure characterization in water (A). Pressure transmitters (PT), flow
sensors (FS), centrifugal pumps (CD) and peristaltic pumps (PP) are
specified with subscripted letters according to their position (R:
Retentate; F: Filtrate; A and AB: Additional sensors on filtrate side;
H: Harvest). Picture of experimental setup (B). Large-scale crossflow
ramping in TFF operation (C) and crossflow ramping with delta pressure
control in HPTFF operation (D). Pressure measurements according to their
position along the filter length are provided for TFF (E) and for HPTFF
(F) at standard operation of 1470 s\textsuperscript{-1} shear, grey
areas represent absolute fiber length.\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
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\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
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\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}\selectlanguage{english}
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\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}\selectlanguage{english}
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\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-4/Figure-4}

\end{center}
\end{figure}\selectlanguage{english}
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\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-5/Figure-5}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-6/Figure-6}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/Figure-7/Figure-7}

\end{center}
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