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\begin{document}

\title{Viral clearance in end-to-end continuous process for mAb purification:
Total flow-through integrated polishing on two columns connected to
virus filtration}



\author[1]{Hironobu Shirataki}%
\author[2]{Yoshihiro Matsumoto}%
\author[3]{Fuminori Konoike}%
\author[4]{Shuichi Yamamoto}%
\affil[1]{Asahi Kasei Medical Kabushiki Kaisha}%
\affil[2]{JNC Kabushiki Kaisha Yokohama Kenkyujo}%
\affil[3]{Kabushiki Kaisha Kaneka Takasago Kenkyujo}%
\affil[4]{Yamaguchi Daigaku Kogakubu}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
There are few reports of the adoption of continuous processes in
bioproduction, particularly the implementation of end-to-end continuous
processes, due to difficulties such as feed adjustment, production batch
demarcation, and incorporating virus filtration. Here, we propose an
end-to-end continuous process for a monoclonal antibody (mAb) with three
integrated process segments: upstream production processes with
pool-less direct connection, pooled low pH virus inactivation with
automated pH control and a total flow-through integrated polishing
process in which two columns were directly connected with a virus
filter. The pooled virus inactivation step demarcates the batch, and
high impurities reduction and mAb recovery were achieved for batches
conducted in succession. Viral clearance tests also confirmed robust
virus reduction for the flow-through two column chromatography and the
virus filtration steps. Additionally, viral clearance tests with two
different hollow fiber virus filters operated at flux ranging from 1.5
to 40 LMH confirmed robust virus reduction over these ranges. Complete
clearance with LRV [?] 4 was achieved even with a process pause at the
lowest flux. The end-to-end continuous process proposed in this study is
highly applicable to production processes, and the investigated virus
filters have excellent applicability to continuous processes conducted
at constant flux.%
\end{abstract}%



\sloppy


\textbf{Viral clearance in end-to-end continuous process for mAb
purification: Total flow-through integrated polishing on two columns
connected to virus filtration}

Hironobu Shirataki\textsuperscript{1}, Yoshihiro
Matsumoto\textsuperscript{2}, Fuminori Konoike\textsuperscript{3},
Shuichi Yamamoto\textsuperscript{4}

\textsuperscript{1}Scientific Affairs Group, Bioprocess Division, Asahi
Kasei Medical Co., Ltd., 1-1-2, Yurakucho, Chiyoda-ku, Tokyo 100-0006,
Japan

\textsuperscript{2}Yokohama R\&D Center, JNC Corporation, 5-1, Ookawa,
Kanazawa-ku, Yokohama-shi, Kanagawa, 236-8605, Japan

\textsuperscript{3}Bio-Pharma Research Laboratories, Kaneka Corporation,
1-8, Miyamae-cho, Takasago-cho, Takasago-shi, Hyogo, 676-8688, Japan

\textsuperscript{4} Yamaguchi University Biomedical Engineering Center
(YUBEC), Yamaguchi University, 2-16-1 Tokiwadai, Ube, Yamaguchi
755-8611, Japan

Corresponding author: Hironobu Shirataki, Scientific Affairs Group,
Bioprocess division, Asahi Kasei Medical Co., Ltd., 1-1-2, Yurakucho,
Chiyoda-ku, Tokyo 100-0006, Japan.

email: shirataki.hb@om.asahi-kasei.co.jp

Co-author email addresses

Yoshihiro Matsumoto: ymatsumoto@jnc-corp.co.jp Fuminori Konoike:
Fuminori.Konoike@kaneka.co.jp

Shuichi Yamamoto: shuichi@yamaguchi-u.ac.jp

Short running title: Viral clearance in continuous mAb process

This research was supported by grant number JP18ae0101056, JP18ae0101057
and JP18ae0101058 from the Japan Agency for Medical Research and
Development (AMED)

Conflict of interest disclosure



\textbf{Abstract}

There are few reports of the adoption of continuous processes in
bioproduction, particularly the implementation of end-to-end continuous
processes, due to difficulties such as feed adjustment, production batch
demarcation, and incorporating virus filtration. Here, we propose an
end-to-end continuous process for a monoclonal antibody (mAb) with three
integrated process segments: upstream production processes with
pool-less direct connection, pooled low pH virus inactivation with
automated pH control and a total flow-through integrated polishing
process in which two columns were directly connected with a virus
filter. The pooled virus inactivation step demarcates the batch, and
high impurities reduction and mAb recovery were achieved for batches
conducted in succession. Viral clearance tests also confirmed robust
virus reduction for the flow-through two column chromatography and the
virus filtration steps. Additionally, viral clearance tests with two
different hollow fiber virus filters operated at flux ranging from 1.5
to 40 LMH confirmed robust virus reduction over these ranges. Complete
clearance with LRV [?] 4 was achieved even with a process pause at the
lowest flux. The end-to-end continuous process proposed in this study is
highly applicable to production processes, and the investigated virus
filters have excellent applicability to continuous processes conducted
at constant flux.

\textbf{Keywords} : continuous process, batch low-pH inactivation, batch
definition, feed adjustment, virus filter, inline spike test, process
pause



\textbf{1. Introduction}

Following the proposal to use continuous processes for monoclonal
antibody (mAb) production (Konstantinov and Cooney, 2015), many studies
had demonstrated benefits such as reduced footprint or cost reduction.
However, even now, there are few examples of its adoption into actual
production. The reasons raised for not adopting this technology include
that downstream processes are not yet developed enough to support
continuous processes compared to the upstream process or that it is very
complicated and difficult to operate each step of the production process
while conducting inline quality control and feed adjustment
(Somasundaram et al., 2018; Gerstweiler et al., 2021; Kumar et al.,
2020). It has been reported that most pharmaceutical manufacturers
adopting continuous processes incorporate batch processes into the
overall process instead of conducting end-to-end continuous processes
(Coffman et al., 2021), and there are also proposals to utilize existing
fed-batch process as part of continuous GMP processes (Ferreira et al.,
2022). There are also reports of concrete proposals for making
continuous processes robust, controllable and practical (David et al.,
2020; Rathore et al., 2021; Coffiman et al., 2021).

One of the greatest expected benefits of the continuous process is its
cost advantage over batch process, and it was shown that combining
continuous processes and single-use technology has a clear cost
advantage, especially for processes with relatively small annual
production volumes (Pollard et al., 2016; Arnold et al., 2018; Hummel et
al., 2019; Farid et al., 2020; Mahal et al., 2021). Furthermore, the
benefits of continuous process contribute not only to cost reduction but
also to PMI (process mass intensity) reduction (Catalda et al., 2020).

While the benefits and challenges of continuous processes have been
extensively examined, there are few reports discussing virus filtration
processes to prevent virus contamination, which is a critical step in
the production process. Virus filtration is not only a critical step in
product safety, but also one of the most costly steps along with media
consumption and Protein A chromatography step (Pollard et al., 2016).
Compared to batch processes with flux of around 50 to 60 LMH (liters per
effective surface area of filter in square meters per hour), David et
al. (2019) reported that flux for a virus filter incorporated into a
continuous process is extremely small at 0.3 LMH but virus logarithmic
reduction value (LRV) of \textgreater{} 4 was still achieved even in
these ultra-low flux conditions and the processing volume per unit
membrane area became larger with lower flux. To verify the robustness of
virus filtration in a long-term continuous process, Lute et al. (2019)
performed continuous filtration over 4 days and showed that virus LRV
\textgreater{} 4 was achieved even at a throughput of 6900
L/m\textsuperscript{2}. Bohonak et al. (2021) investigated a continuous
mAb process with a connected column process and virus filter operated in
constant pressure mode, and they reported that the intensified process
in which the mAb solution was concentrated by single-pass tangential
flow filtration (TFF) before the column process showed improved
purification efficiency of mAb and reduced virus filter membrane area
usage. Coolbaugh et al. (2021) achieved continuous operation for 25 days
for a virus filtration process operating in TFF mode incorporated into
an end-to-end integrated and continuous process.

Validation of the virus filter incorporated into a continuous process
cannot be evaluated individually, unlike in a batch process; rather, it
is necessary to consider an appropriate virus validation method for
continuous process. Proposals for various inline virus spiking methods
adapted for virus validation in a continuous process and confirmation of
robust virus removal by these methods has been reported by Lute et al.
(2011), Bohonak et al. (2021), Shirataki et al. (2021a) and Malakian et
al. (2022).

One of the major issues in adopting continuous processes for commercial
production is the definition of the product batch, and ICH Q13 Guideline
(2021) describe that the product batch should be clearly defined as
quantity of output material, quantity of input material and run time at
a defined mass flow rate. Use of the greatest common divisor (GCD) of
the time required for each step of the process has been reported to be
an appropriate definition of batch that meets this requirement (Lali et
al., 2021).

In this study, we present an end-to-end continuous process incorporating
a constant flux filtration process for the virus filter with feed
adjustment between each step and a clear batch concept of the product.
Here, the batch is set as the unit of product pooled in the low pH
inactivation step. In this process, the solution harvested from the
perfusion cell culture is continuously supplied to the affinity process
with Protein A column, and column eluate is pooled for the virus
inactivation step of low pH treatment for 1 h in an automated manner.
After low pH inactivation, the pH-adjusted mAb solution is supplied to a
total flow-through two column polishing process including direct
connection to a virus filter. The combination of the AEX and CEX columns
used here with the virus filter has already been reported to show very
high impurity removal, recovery rate and filterability of the virus
filter in a total flow-through process (Shirataki et al., 2021a). Though
activated carbon is reported to be effective for impurity and virus
clearance by flow-through (Ichikawa et al., 2019; Kikuchi et al., 2022),
we adopted flow-through two column chromatography in this study.

The total flow-through integrated polishing process has been reported to
achieve high mAb purification and robust virus reduction (Shirataki et
al., 2021a). In this report, we verify the effectiveness of this
integrated process when incorporated into a practical scaled-down
end-to-end continuous process. In our previous report (Shirataki et al.
2021a), robust reduction of minute virus of mice (MVM) and xenotropic
murine leukemia virus (X-MuLV) using inline spiking was confirmed for a
mAb process with throughput to about 100 L/m\textsuperscript{2} and low
flux levels expected in the continuous process (5 LMH lowest). In this
report, we examined MVM removal in a mAb solution on two different
hollow fiber virus filters (Planov BioEX and S20N, Asahi Kasei Medical)
to a throughput of about 1000 L/m\textsuperscript{2} and flux of 5 LMH
down to 1.5 LMH with and without process pause to especially test the
effect of process pause in low flux filtration. To test a process with
reduced filterability, we conducted spiking with X-MuLV at increased
titer and compared the performance of two types of hollow fiber virus
filters.

\textbf{2. Materials and Methods}

\textbf{2.1. End-to-end continuous processing}

The schematic and process diagram of the end-to-end continuous process
from cell culture to purification that was used in this study is shown
in Figure 1. In the process diagram, washing, equilibration,
regeneration and replacement of columns and filters before and after
each step are shown in gray. Starting from day 12 of cell culture, mAb
expressed by the perfusion cell culture was harvested and purified by
successively performing Protein A column capture, pooled low pH virus
inactivation, and flow-through two column chromatography and virus
filtration (total flow-through integrated polishing). In the end-to-end
continuous process used in this study, the process stream was only
pooled for the low pH virus inactivation process, and this production
unit is used to demarcate the batch. In this study, a total of three
batches were processed in succession. Impurities and mAb concentration
at each step were evaluated off-line (Figure 2).

For the end-to-end continuous process trials, mAb (pI 8.5) expressed by
a CHO-MK cell line in perfusion cell culture developed by the
Manufacturing Technology Association of Biologics (MAB) at its GMP
facility was used. The proprietary process consists of harvest of the
perfusion cell culture process by XCell ATF-2 (Repligen) at 1.1 vvd
(vessel volume exchanges per day), followed by a continuous capture
process on two 1-mL Protein A columns (KANEKA KanCapA 3G, Kaneka
Corporation) in periodic counter current (PCC) mode on a Contichrom HPLC
30 (YMC Co., Ltd.) with continuous elution of purified mAb with 60 mM
acetate (pH 3.6) buffer in cycles of total 20-22 h, with pooling of the
eluate for each cycle and column regeneration between total cycles. Each
batch of pooled eluate was automatically adjusted to pH 3.4, held for 60
min, then automatically adjusted to pH 6.5, 10 mS/cm with 1 M Tris
buffer and 1 M NaCl, and filtered with a micro filter with a nominal
pore size of 0.1 \selectlanguage{greek}µ\selectlanguage{english}m (USP-043, Asahi Kasei Co., Ltd.) to remove
precipitates. The process solution was supplied to the polishing process
with two columns directly connected to a virus filter at a constant flow
rate.

The total flow-through integrated polishing process consisted of two
prepacked mini-columns of AEX (Cellufine MAX IB) and CEX (Cellufine MAX
DexS-HbP; both Mini-Column 5LM; 14.6 mm ID, 30 mm L; CV of 5 mL; JNC
Corporation) and a Planova BioEX virus filter (0.0003
m\textsuperscript{2}; Asahi Kasei Medical) without pooling between each
step, and the final purified mAb solution was collected. For the
polishing chromatography steps, the AEX and CEX columns were directly
connected in series to column valves of the AKTA pure 25 (Cytiva),
equilibrated with 20 mM Tris-acetate, pH 6.5, 10 mS/cm, which was used
for purification of mAb. The virus filter was connected to the outlet
valve of the AKTA pure 25 and prepared for use independently of the
columns. The AKTA pure 25 sample pump was operated at constant flow
rate, and the virus filter inlet pressure (Figure 3) was recorded with a
pressure sensor PREPS-N-000 (PendoTECH). Host cell proteins (HCP), host
cell DNA and mAb concentrations and aggregates ratio were analyzed at
each step of each batch by the following methods.

\textbf{mAb concentration}

mAb concentration (mg/mL) was determined in cell culture by analytical
protein A-HPLC performed on a chromatography system consisting of MU701
UV-VIS detector and a PU7710 pump ( GL Sciences, Inc.) using a silica
monolith Protein A column (Kyoto Monotech, Co., Ltd.) and for process
solutions by bind and elute chromatography mode using 50 mM phosphate
buffer with 1 M NaCl, pH 7.0 as the mobile wash phase and 100 mM sodium
citrate, pH 3.5 as the mobile elution phase followed by quantification
of mAb in purified solution by absorption measurement at 280 nm on a
UV-VIS spectrophotometer V-750 (JASCO Corporation).

\textbf{HCP concentration}

CHO host cell protein (HCP) concentration (ng/mg-mAb) was determined
using the CHO HCP 3rd generation ELISA kit (F550) (Cygnus Technologies)
according to manufacturer instructions.

\textbf{Host cell DNA concentration}

Host cell DNA concentration (pg/mg-mAb) was determined by quantitative
PCR (qPCR) performed using the PrepSEQ Residual DNA Sample Preparation
Kit and resDNASEQ Quantitative CHO DNA Kit (Thermo Fisher Scientific,
Inc.) according to manufacturer protocol.

\textbf{mAb aggregates concentration}

mAb aggregate concentration (\% in mAb) was determined by analytical
size exclusion chromatography (SEC-HPLC) performed on a chromatography
system Prominence LC-20 (Shimadzu Corporation) using a TSKgel G3000SWxl
5 \selectlanguage{greek}μ\selectlanguage{english}m column (Tosoh Bioscience, LLC) with a mobile phase of 100 mM
potassium phosphate with 200 mM NaCl, pH 7.0.

\textbf{2.2. Viral clearance studies}

Viral clearance studies on various components of the flow-through two
column chromatography (AEX and CEX) and virus removal filter were
conducted at ViruSure (Vienna, Austria) using purified mAb produced from
the same cell line but by fed-batch production and purification with
Protein A and IEX columns. The mAb used in these studies was 10 mg/mL
mAb in 20 mM Tris-acetate, 10 mS/cm, pH 6.5. MVM and X-MuLV virus titers
were determined by TCID\textsubscript{50} method.

\textbf{2.2.1. Flow-through two column chromatography}

The virus removal capability of the Cellufine MAX IB (AEX) and Cellufine
DexS HbP (CEX) mini columns (CV of 5 mL) operated in flow-through mode
without virus filtration (Figure 4) was evaluated. Following column
equilibration, 300 mL of 10 mg/mL mAb (600 mg mAb/mL-resin for each
column) spiked with MVM or X-MuLV at 2\% and passed through a 0.45 \selectlanguage{greek}μ\selectlanguage{english}m
PES prefilter was loaded onto the columns at a flow rate of 0.25 mL/min
(residence time 20 min) using an AKTA pure 25. Following a 60 min
process pause, the columns were flushed with a 30 mL buffer wash. Virus
titer assay was conducted on both the 300 mL flow-through fraction and
the combined flow-through fraction with the 30 mL buffer wash (Table 1).

\textbf{2.2.2. Virus filter in total flow-through integrated polishing
process (inline spike test)}

To confirm the virus removability of the virus filter in the total
flow-through integrated polishing process shown in Figure 5, the inline
spiking viral clearance study method designed by ViruSure as reported by
Shirataki et. al. (2021a) was conducted. For this study, 10 mg/mL mAb
spiked at 10\% with one of two viruses, MVM or X-MuLV, was passed
through a 0.1 \selectlanguage{greek}μ\selectlanguage{english}m PES prefilter or 0.2 \selectlanguage{greek}μ\selectlanguage{english}m PES prefilter, respectively
before use. The flow-through two column chromatography process described
in Section 2.2.1 was directly connected without pooling to a 0.0003
m\textsuperscript{2} Planova BioEX filter on an AKTA pure 25. Un-spiked
10 mg/mL mAb was supplied to the two column system by system pump at a
flow rate of 0.18 mL/min while the 10\% virus (MVM or X-MuLV) spiked mAb
was supplied by sample pump at a flow rate of 0.02 mL/min to the feed
stream after the two chromatography columns and before the virus filter,
resulting in mAb with 1\% virus spike being applied to the filter at a
constant flow rate of 0.2 mL/min, corresponding a flux of 40 LMH. After
loading 300 mL of mAb solution, both pumps were stopped for a 60 min
process pause. Then, only the system pump was turned on to supply 30 mL
of buffer at a flow rate of 0.2 mL/min to wash out feed from the
chromatography columns. Using this procedure, viral clearance was
conducted at a constant flow rate and the effect of a process pause was
evaluated (Table 2). The total mAb solution loaded to virus filter was
300 mL and loading of mAb on the filter by effective surface area was
about 10 kg/m\textsuperscript{2} and 1000 L/m\textsuperscript{2}.

\textbf{2.2.3. MVM spike of low flux filtration on Planova BioEX and
S20N filters}

Viral clearance studies of the mAb were conducted on 0.001 and 0.0003
m\textsuperscript{2} Planova BioEX filters, which has a hydrophilic
modified polyvinyldene flouride (PVDF) hollow fiber, and on the 0.001
m\textsuperscript{2} Planova S20N filter (Asahi Kasei Medical), which is
a newly launched filter with hollow fibers made from regenerated
cellulose. In this study, all runs were conducted at low flux of 1.5, 5
and 10 LMH for Planova BioEX and 1.5 LMH for Planova S20N as shown in
Table 3. For all runs, 10 mg/mL mAb spiked with 1\% MVM (0.1 \selectlanguage{greek}μ\selectlanguage{english}m
prefiltered) was filtered for 24 h. The spike solution was loaded to the
filter using the sample pump of AKTA pure 25. Following mAb load
filtration and a 60 min process pause, 10 mL or 5 mL of equilibration
buffer wash was conducted for the 0.001 m\textsuperscript{2} or 0.0003
m\textsuperscript{2} filters, respectively. Viral clearance was measured
for the mAb load permeate and total permeate with buffer wash.

\textbf{2.2.4. Higher spike X-MuLV on Planova BioEX and S20N filters}

To conduct viral clearance with a higher load titer of X-MuLV, 10 mg/mL
mAB spiked with 2\% X-MuLV (0.2 \selectlanguage{greek}μ\selectlanguage{english}m prefiltered) was filtered on 0.0003
m\textsuperscript{2} Planova BioEX and 0.001 m\textsuperscript{2} S20N
filters with a load volume of 300 and 1000 mL, respectively,
corresponding to a throughput of 1000 L/m\textsuperscript{2}, and a flow
rate of 0.2 and 0.667 mL/min, respectively, corresponding to a flux of
40 LMH for each filter (Table 4). The spike solution was loaded to the
filter using the sample pump of AKTA pure 25. The log titer of load
solution in this test is effectively higher than the inline spike test
described previously because of higher spike concentration.

\textbf{3. Results and Discussion}

\textbf{3.1. End-to-end continuous process with total flow-through
integrated polishing process}

In this end-to-end continuous process, the mAb solution produced by
perfusion cell culture was clarified by ATF and purified by Protein A
chromatography all with pool-less direct connection. The obtained
material was pooled for low pH virus inactivation, and this volume was
taken as the batch. Finally, total flow-through integrated polishing was
conducted in a pool-less direct connection as shown in Figure 1.

\textbf{3.1.1. Performance at each step of end-to-end continuous process
for mAb purification}

Process performance was examined for the end-to-end continuous process
incorporating upstream production processes with pool-less direct
connection, pooled low pH virus inactivation with automated pH control
and a total flow-through integrated polishing process in which two
column chromatography was directly connected to a virus filter. The flow
rates through the process were set to accommodate completion of batches
in succession to allow continuously utilizing the setups (Figure 1).
Batch 1 had a smaller volume but higher mAb concentration supplied to
the polishing step of 116.14 mL and 7.6 mg/mL (Figure 3), respectively,
due to air bubbles impeding the supply of harvest solution to the
capture process. For subsequent batches, the load volume and mAb
concentration were 315.29 mL and 5.1 mg/mL for Batch 2, and 317.97 mL
and 4.4 mg/mL for Batch 3, respectively. The pH- and
conductivity-adjusted mAb solution after the low pH virus inactivation
step was supplied to the flow-through two column chromatography
consisting of AEX and CEX columns directly connected to the virus filter
at a flow rate of 0.14 mL/min for Batch 1 and 0.38 mL/min for Batches 2
and 3. The flow rate were determined to adjust the batch cycle time as
about 24 hours. At these flow rates, the process feed had a residence
time for each column of 35.7 min for Batch 1 and 13.2 min for Batches 2
and 3. The mAb throughput for each column was 176.5 mg/mL-resin for
Batch 1, 321.6 mg/mL-resin for Batch 2, and 280.7 mg/mL-resin for Batch
3. At the end of applying the mAb to the flow-through two column
process, equilibration buffer (15 mL for Batch 1 and 75 mL for Batches 2
and 3) was applied to wash out residual mAb in the columns.

As the flow-through from the columns was directly loaded to the virus
filter, the filtration was conducted at the same constant flow rate as
the polishing chromatography step and the filtrate fraction was
consisting of the flow-through fraction followed by the buffer wash
fraction. At first, the filtration pressure of the Planova BioEX plotted
against filtration time (min) and throughput (L/m\textsuperscript{2})
shows pressure of about 0.073 MPa for Batch 1 and about 0.160 MPa for
Batches 2 and 3 for the filtration of equilibration buffer (Figure 3).
The pressure increased as mAb entered the filter, remained stable and
higher (about 0.077 MPa for Batch 1, about 0.175 MPa for Batch 2 and
about 0.170 MPa for Batch 3) during the flow-through mAb filtration,
then following a partial depressurization lasting several seconds due to
flow path switching, the filtration pressure decreased again. For
Batches 1 and 3, pressure fluctuation occurred during filtration of the
wash buffer, and this may be due to unstable supply of buffer from the
pump and introduction of air during path switching. Batch 2 showed a
slightly higher filtration pressure than Batch 3 due to a slightly
higher mAb concentration of 5.10 mg/mL compared to 4.41 mg/mL for Batch
3. For all batches, the filtration pressure remained almost constant
with almost no increase, even for throughput of more than 1000
L/m\textsuperscript{2} for Batches 2 and 3. The flow-through two column
chromatography process effectively removes impurities such as HCP and
aggregates (Figure 2), resulting in a very highly purified mAb solution
with high filterability. This demonstrates that the total flow-through
integrated polishing process adopted in the end-to-end continuous
process of this study is extremely effective for purification and for
filterability for virus removal.

Focusing on the virus filter performance, the flow rate through the
filter matches the flow rate through the columns and was 28 LMH for
Batch 1 and 76 LMH for Batches 2 and 3, and by volume-based throughput
(L/m\textsuperscript{2}) and mass-based throughput
(kg/m\textsuperscript{2}), the mAb throughput was 387.1
L/m\textsuperscript{2} and 2.9 kg/m\textsuperscript{2} for Batch 1, 1051
L/m\textsuperscript{2} and 5.4 kg/m\textsuperscript{2} for Batch 2, and
1059.9 L/m\textsuperscript{2} and 4.7 kg/m\textsuperscript{2} for Batch
3. In addition, the total filtrate volume including the wash from the
column, was 131.1 mL for Batch 1, 391.8 mL for Batch 2, and 393.9 mL for
Batch 3, and throughput for the filter including wash was 437.1
L/m\textsuperscript{2}, 1301.0 L/m\textsuperscript{2} and 1309.9
L/m\textsuperscript{2}, respectively for each batch.

\textbf{3.1.2. mAb purification with impurity removal}

The impurity levels and mAb concentration are shown in Figure 2. For all
three batches, the concentrations of mAb and impurities showed a similar
trend for all steps. The mAb concentration increased in the capture step
and decreased in the subsequent low pH virus inactivation and polishing
steps due to volume increases accompanying pH and conductivity
adjustment and the addition of wash buffer, resulting in 3.4 - 5.2 mg/mL
mAb after the polishing step including virus filter. The mAb recovery
for the total flow-through integrated polishing step was 77\%, 93\% and
95\% for Batches 1, 2 and 3, respectively. The lower recovery rate for
Batch 1 was likely due to using less buffer volume for wash and
increasing the volume from 15 to 75 mL produced a recovery rate of more
than 90\%.

Impurities were overall reduced by the process. The HCP concentration
exponentially decreased at each process step, especially in the capture
and polishing steps, decreasing to 12.0 ng/mg-mAb or less. The DNA
concentration also decreased exponentially, particularly in the capture
and low pH virus inactivation steps, reaching 1 pg/mg-mAb or less after
the polishing step. The aggregate concentration was around 1\% in the
harvest step, increased to around 3\% in the capture and low pH virus
inactivation steps, and then decreasing to less than 1\% after the
polishing step. High aggregate content was produced by the long-term
(20-22 h) exposure of mAb to low pH in the capture elution pool,
although the KANEKA KanCapA\textsuperscript{TM} 3G Protein A columns can
efficiently reduce aggregates (Pabst et al., 2018). SEC analysis of the
process solution confirmed that the aggregates were primarily dimers,
and trimers or higher aggregates were below the detection limit. Since
the virus filter removes virus particles by size exclusion, it is
thought to not remove small-sized HCP and dimers. The HCP and aggregate
contents were reduced by the polishing step as shown in Figure 2,
corroborating that the flow-through two column chromatography shows
excellent HCP and aggregate removability as reported previously
(Shirataki et. al., 2021a; 2021b).

\textbf{3.2. Virus removability of total flow-through integrated
polishing process}

In the end-to-end continuous process for mAb purification, virus removal
is expected for the Protein A capture, low pH virus inactivation, two
column chromatography and virus filter steps, with the most robust virus
removability at the virus filtration step. Here, viral clearance testes
were carried out for specific segments of the total flow-through
integrated polishing process.

\textbf{3.2.1. Viral clearance for flow-through two column
chromatography}

Previously, the virus clearance test for individual column (AEX and CEX)
and the inline virus spiking test for virus filter directly connected to
a column, showed excellent virus removability as reported (Shirataki et
al., 2021a). However, in that report, the residence time for the column
was as short as 2 min compared to 13 to 35 min in this study, and each
column was evaluated individually with throughput of 300 mg mAb
/mL-resin. In this study, virus removability was examined for the
flow-through two column chromatography with a residence time of 20 min
and a mAb throughput of 600 mg/mL-resin for each column.

For the flow-through two column chromatography system with AEX and CEX,
the UV absorbance profile of the MVM-spiked mAb solution run shows that
buffer in the columns is rapidly replaced by mAb solution as indicated
by the UV absorbance sharply rising and remaining at the maximum level
throughout the run (Figure 4). Following application of the specified
volume of solution to the two columns and a 60 min process pause, the
buffer wash cleared the mAb from the columns as shown by the gradually
decreasing UV absorbance. As shown in Table 1 for mAb spiked with MVM or
X-MuLV, complete clearance was achieved for both the flow-through
fraction and the flow-through plus wash with LRV of [?] 4 for MVM and
X-MuLV. These results confirm that the two column chromatography has
high virus removability for both X-MuLV and MVM for a process with
residence time of 20 min in the columns and that process pause did not
affect virus removal.

\textbf{3.2.2. Inline spiking viral clearance test for integrated
flow-through two column and virus filter}

To separately examine the virus removability of the virus filter, an
inline spiking viral clearance test for the Planova BioEX filter
implemented in the flow-through two column chromatography was carried
out. Due to clogging from impurities in the virus spike, the flow rate
was reduced to produce a flux of 40 LMH to the filter (compared to 76
LMH for the end-to-end continuous process) to avoid exceeding the
operating pressure limit of Planova BioEX (0.35 MPa) before reaching the
target throughput. It should be noted that viral clearance determined
for the lower flux process (40 LMH) can be taken as confirming viral
clearance at 76 LMH, as lower flux is a worse case condition for virus
filtration.

As shown in Figure 5, a feed solution of 10 mg/mL mAb without virus
spike was supplied to the two columns and the same mAb spiked at 10\%
MVM or X-MuLV was supplied through inline spiking. The pressure
increases on the filter were observed until the peak after the process
pause for the loading of mAb solution spiked with MVM or X-MuLV compared
to the minimal pressure increase observed for loading of mAb solution
without spike. The pressure profile of the virus-spiked mAb filtration
shows fluctuations that are considered to be due to pulsation of the
pump due to air bubbles. The pressure increases with increasing load
volume are greater for the filtration of the X-MuLV spiked solution and
approach the upper operating pressure limit of the filter at the target
throughput. The more stable filtration pressure of the un-spiked mAb is
similar to the pressure profile for the filter incorporated in the
end-to-end continuous process shown in Figure 3.

Due to the concern that virus breakthrough to permeate may occur due to
the pressure drop during a process pause as previously described (Ajayi
et al., 2022; Fan et al., 2021; Johnson et al., 2021), a 60 min process
pause was included after loading the virus-spiked mAb, and virus
removability for inline spiking of MVM or X-MuLV was evaluated for both
the flow-through fraction of the mAb and the flow-through fraction with
the buffer wash. As shown in Table 2 for mAb spiked with MVM or X-MuLV,
complete clearance was achieved for both the flow-through fraction and
the flow-through plus wash with MVM LRV of [?] 5 and X-MuLV LRV of [?] 3.
The relatively lower X-MuLV LRV for the filter than the two column
process can be attributed to the lower X-MuLV spike titer. These results
confirm that the Planova BioEX filter has high virus removability for
both MVM and X-MuLV and that the process pause did not affect virus
removal.

\textbf{3.2.3. Viral clearance at low flux for Planova BioEX and Planova
S20N}

For 24 h filtrations of 10 mg/mL mAb spiked with MVM on two sizes of
Planova BioEX and one size of Planova S20N to test low flux (1.5 - 10
LMH) conditions, complete MVM removal was achieved for all load and load
plus wash buffer as shown in Table 3. For the load fraction as well as
for the total load fraction plus buffer wash after the 60 min process
pause, MVM LRV of \textgreater{}5 was confirmed for all runs. Under
these low flux conditions, filtration pressure was extremely low, about
0.04 MPa at 5 LMH for Planova BioEX, and at 1.5 LMH, pressure was less
than 0.01 MPa for both Planova BioEX and S20N. These results confirm
that both Planova BioEX and S20N filters have robust virus removal
properties, even for very low flux filtrations with process pause, such
as in continuous process applications.

\textbf{3.2.4. Viral clearance with higher spike concentration}

As shown in the viral clearance study in the integrated process (Table
2), the load titer of X-MuLV is sometimes lower than that for MVM even
with the same spike level. Lower load titer may cause a lower virus LRV
than expected, and in this case, X-MuLV LRV of \textgreater{} 4 could
not be achieved, despite that no virus was detected in the permeate,
indicating complete clearance. The lower spike titer for X-MuLV may have
been due to the instability of X-MuLV, and similar results have been
reported previously (Shirataki et al., 2021a; Gefroh et al., 2014).

To test virus removal for a higher spike level, 10 mg/mL mAb spiked at
2\% with X-MuLV was processed to virus filter at 40 LMH with a 60 min
process pause. As shown in Table 4, much higher X-MuLV titer was
achieved by increasing the virus spike to 2\% (6.69 and 7.27 log
TCID\textsubscript{50}/mL for Planova BioEX and S20N, respectively)
compared to 5.84 log TCID\textsubscript{50}/mL for the 1\% spike (Table
2). An X-MuLV LRV \textgreater{} 4 was achieved for both Planova BioEX
and S20N filters with complete clearance for both the flow-through
fraction and the flow-through fraction plus wash. Thus, the reason
X-MuLV LRV did not exceed 4 in Table 2 and in the previous report
(Shirataki et al., 2021a) was that the load titer was not sufficient.

\textbf{4. Conclusions}

The end-to-end continuous process for mAb purification reported in this
study has upstream and downstream steps with pool-less direct
connections and a low pH inactivation step with automated pH adjustment
performed in batch mode, which serves as a batch demarcation and also
allows for feed adjustment before and after the low pH inactivation
step. These characteristic properties make the process amenable for
continuous mAb production. Furthermore, the mAb production experiment by
the scale-down model of this report confirmed that this continuous
process results in high productivity and high purity of mAb. The mAb was
highly purified by the total flow-through integrated polishing process
in which two columns (AEX and CEX) were directly connected to the virus
filter. As a result, even with a filtration throughput of about 1000
L/m\textsuperscript{2}, filtration pressure increases on the virus
filter were extremely small, demonstrating that high purification and
extremely stable virus filtration can be realized in the end-to-end
continuous process for mAb purification.

Regarding virus reduction, both the flow-through two column
chromatography and the virus filter showed robust virus removability for
the total flow-through integrated polishing process. While virus
filtration is usually carried out by constant pressure filtration and is
not easily incorporated into a continuous process, the hollow fiber type
virus filters tested in this study showed high virus reduction even at
very low flux and with a process pause that may be observed in
continuous processing. For very low flux of 1.5 LMH and a condition with
process pause of 60 min, both Planova BioEX and S20N showed virus LRV of
\textgreater{} 4, indicating that these hollow fiber type virus filters
are highly applicable for the continuous process with high filterability
and robust virus removability.

\textbf{Acknowledgements}

The authors thank Linda Gudex and Elizabeth Nguyen (Asahi Kasei
Bioprocess America) for their assistance with manuscript preparation.
This research was supported by grant number JP18ae0101056, JP18ae0101057
and JP18ae0101058 from the Japan Agency for Medical Research and
Development (AMED)

\textbf{Conflict of Interests}

The authors (HS, HM and HK) are employees of their affiliate companies.
None have received any compensation for the research reported in this
article beyond that from their affiliate employee.

\textbf{Author Contribution}

Hironobu Shirataki: Conceptualization, Data curation, Formal analysis,
Investigation, Writing - original draft, Writing - Review \& Editing.
Yoshihiro Matsumoto: Data curation, Formal analysis. Fuminori Konoike:
Data curation, Formal analysis. Shuichi Yamamoto: Conceptualization,
Supervision, Writing - original draft.

\textbf{Data Availability Statement}

The data that support the findings of this study are available on
request from the corresponding author. The data are not publicly
available due to privacy or ethical restrictions.

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\textbf{Figure legends}

\textbf{Figure 1} . Schematic and process diagram representing
end-to-end continuous process for mAb production with implementation of
total flow-through integrated polishing process

\textbf{Figure 2} . Changes throughout mAb harvest, capture, low pH
virus inactivation and polishing processes for a) HCP (ng/mg-mAb), b)
DNA (pg/mg-mAb), c) mAb (mg/mL) and d) aggregate (\%).

\textbf{Figure 3} . Transmembrane pressure of mAb virus filtration
flow-through fraction and buffer wash implemented in the end-to-end
continuous process shown in Figure 1 for three batches with a) pressure
plotted against time (min) and b) pressure plotted against throughput
(L/m\textsuperscript{2}).

\textbf{Figure 4} . Setup of viral clearance test for flow-through two
column chromatography (upper) and 280 nm UV absorbance profile of
flow-through output for MVM-spiked mAb run (lower).

\textbf{Figure 5} . Setup of inline spiking viral clearance test for
integrated polishing process with virus filter connected to flow-through
two columns chromatography (upper figure) and transmembrane pressure
profile of virus filter loaded with virus-spiked mAb flow-through from
the two column step (lower figure).

\textbf{Tables}

\textbf{Table 1} . Viral clearance test results for flow-through two
column chromatography consisting of Cellufine MAX IB and Cellufine DexS
HbP with 10 mg/mL mAb spiked with MVM and X-MuLV.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\begin{minipage}[b]{0.32\columnwidth}\raggedright\strut
Parameter\strut
\end{minipage} & \begin{minipage}[b]{0.32\columnwidth}\raggedright\strut
MVM\strut
\end{minipage} & \begin{minipage}[b]{0.32\columnwidth}\raggedright\strut
X-MuLV\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Load titer (log TCID\textsubscript{50}/mL)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
8.93\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
6.29\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Flow-through fraction virus LRV\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
[?]6.34~\selectlanguage{ngerman}±~0.18\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
[?]4.34~\selectlanguage{ngerman}±~0.25\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Flow-through + Wash virus LRV\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
[?]6.07~\selectlanguage{ngerman}±~0.18\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
[?]4.06~\selectlanguage{ngerman}±~0.25\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\textbf{Table 2} . Virus reduction of inline spike virus clearance test
for Planova BioEX implemented in the total flow-through integrated
polishing process with 10 mg/mL mAb. Spiked virus; MVM and X-MuLV.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Parameter & MVM & X-MuLV\tabularnewline
Load virus spike (log TCID\textsubscript{50}/mL) & 7.54 &
5.84\tabularnewline
Flow-through fraction virus LRV (log TCID\textsubscript{50}/mL) &
[?]5.47~\selectlanguage{ngerman}±~0.25 & [?]3.78~\selectlanguage{ngerman}±~0.25\tabularnewline
Flow-through + Wash virus LRV (log TCID\textsubscript{50}/mL) &
[?]5.07~\selectlanguage{ngerman}±~0.24 & [?]3.37~\selectlanguage{ngerman}±~0.25\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\textbf{Table 3} . Viral clearance test results with low flux loading on
Planova BioEX and S20N for 10 mg/mL mAb spiked with MVM.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Parameter\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Filtration runs\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Filtration runs\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Filtration runs\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Filtration runs\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Filter\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Planova BioEX 0.001 m\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Planova S20N 0.001 m\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Planova BioEX 0.0003 m\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Planova BioEX 0.0003 m\textsuperscript{2}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Flow rate (mL/min)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.025\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.025\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.025\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.05\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Flux (LMH)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.5\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.5\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
10\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Load (mL)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
36\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
36\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
36\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
72\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Throughput (L/m\textsuperscript{2})\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
36\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
36\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
120\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
240\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
MVM spike (log TCID\textsubscript{50}/mL)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
7.71\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
7.86\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
7.41\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
7.96\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Flow-through fraction MVM LRV (log TCID\textsubscript{50}/mL)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
[?]6.04~\selectlanguage{ngerman}±~0.26\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
[?]6.30~\selectlanguage{ngerman}±~0.28\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
[?]5.79~\selectlanguage{ngerman}±~0.28\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
[?]6.03~\selectlanguage{ngerman}±~0.27\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Flow-through fraction + Wash MVM LRV (log
TCID\textsubscript{50}/mL)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
5.43~\selectlanguage{ngerman}±~0.45\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
[?]5.91~\selectlanguage{ngerman}±~0.28\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
[?]5.64~\selectlanguage{ngerman}±~0.28\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
[?]6.15~\selectlanguage{ngerman}±~0.27\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\textbf{Table 4} . Viral clearance test results for Planova BioEX and
S20N with 10 mg/mL mAb spiked with 2\% X-MuLV.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Parameter & Filtration runs & Filtration runs\tabularnewline
Filter & BioEX 0.0003 m\textsuperscript{2} & S20N 0.001
m\textsuperscript{2}\tabularnewline
Flow rate (mL/min) & 0.2 & 0.667\tabularnewline
Flux (LMH) & 40 & 40\tabularnewline
Load (mL) & 300 & 1000\tabularnewline
Throughput (L/m\textsuperscript{2}) & 1000 & 1000\tabularnewline
X-MuLV load (log TCID\textsubscript{50}/mL) & 6.69 & 7.27\tabularnewline
Flow-through fraction X-MuLV LRV (log TCID\textsubscript{50}/mL) & [?]4.59
\selectlanguage{ngerman}± 0.28 & [?]4.65 \selectlanguage{ngerman}± 0.27\tabularnewline
Flow-through fraction + Wash X-MuLV LRV (log TCID\textsubscript{50}/mL)
& [?]4.53 \selectlanguage{ngerman}± 0.28 & [?]4.63 \selectlanguage{ngerman}± 0.27\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

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\selectlanguage{english}
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