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‐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‐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/m2/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.