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\begin{document}

\title{Protection of Biomanufacturing Processes from Virus Contamination
through Upstream Virus Filtration of Cell Culture Media}



\author[1]{Andreas Wieser}%
\author[1]{Jens Modrof}%
\author[1]{Thomas Kreil}%
\affil[1]{Takeda Manufacturing Austria AG}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Medical progress as enabled by early plasma products has also revealed
biological safety challenges. The combination of donor selection,
donation testing and virus reduction processes has effectively addressed
these concerns, and today medicinal plasma products feature significant
safety margins. The safety tripod concept has since been adapted to
biotechnology manufacturing platforms and has also ensured the safety of
these products. However, cell-based manufacturing processes have
occasionally been exposed to adventitious viruses, leading to
manufacturing interruptions and unstable supply situations. The rapid
progress of advanced therapy medicinal products (ATMPs) needs an
innovative approach to ensure the learnings from more traditional
biotechnology help to avoid any unwelcome reminder of the universal
presence of viruses. The introduction of up-stream virus clearance steps
has already been shown to be valuable for any products too complex for
down-stream interventions in the sense of both assuring product safety
and continuous supply. The gentlest method being virus filtration -- the
development of which is presented here. The experiments investigated the
feasibility of implementing culture media virus filtration with respect
to their virus clearance capacities under extreme conditions such as
very high process feed loading (up to \textasciitilde{} 19,000 L/m
\textsuperscript{2}), very long duration (up to 31 days), and multiple
process interruptions (up to 21, with cumulative interruptions of over
92 hours). Minute virus of mice (MMV) was used as a relevant target
virus, and in general, as a model small non-enveloped virus, as these
viruses are the main challenge for the investigated virus filters with a
stipulated pore-size of about 20 nm. It was found that certain filters
-- especially of the newer 2 \textsuperscript{nd} generation -- are
capable of effective virus clearance despite the harsh regimen they were
subjected to. At the same time the investigation of biochemical
parameters for un-spiked control runs showed the filters to have no
measurable impact on the composition of the culture media. Based on
these findings, this technology seems to be quite feasible for large
volume pre-manufacturing process culture media preparations.%
\end{abstract}%



\sloppy


\textbf{Introduction}

The production processes of biopharmaceutical products based on
eucaryotic expression systems carry an intrinsic risk for viral
contaminations. An industry wide data collection of such contamination
events revealed that contamination sources were traced primarily to raw
materials or cell culture media including specific components thereof,
like FBS (Barone, 2020).

Traditionally, to mitigate this risk, the safety tripod concept (Kreil,
2018) is applied; (a) raw materials used in manufacturing of
biopharmaceuticals are selected for low risk, under consideration of
viral safety aspects, (b) raw materials are tested for potential viral
contaminants, and most importantly the main proportion of safety margins
is achieved through (c) virus inactivation or removal steps integrated
in the ``down-stream'' manufacturing process. These steps are either
dedicated virus clearance steps, or steps with an intrinsic capacity to
clear viruses. As required by current regulations (EMEA 1996; ICH, 1999,
FDA, 1997), dedicated virus clearance steps are integrated into
manufacturing processes to increase the safety margins of
biopharmaceuticals, in a well-controlled manner.

However, for active (``live'') biopharmaceuticals, such as live virus
vaccines, or Advanced Therapy Medicinal Products (ATMPs) like cellular
therapies and gene therapies, dedicated viral clearance procedures
cannot always be applied to the down-stream processes as they would not
only inactivate or remove potentially present viral contaminations but
equally compromise activity of the biopharmaceutical ingredient.

This is especially true for virus filtration, one of the most robust and
effective viral clearance procedures that can conceptually remove all
pathogens larger than the stipulated pore-size of the filter.
Unfortunately, for most live virus vaccines or ATMPs the therapeutic
biomolecule is also larger, and thus effective virus clearance is not
possible.

Furthermore, some large biomolecules, such as von Willebrand Factor
(VWF) complexes cannot pass through the smallest type of virus filters
which are suitable for the removal of e.g. Parvovirus without
significant losses or severe clogging events due to the nature of the
molecules (Parker, 2021). In these cases, the introduction of
down-stream virus filtration as dedicated virus clearance step is
technically impossible.

However, taking specifically the main source of virus risk for cell
culture processes into account, the virus filtration procedure can be
directly applied to mitigating that risk by filtration of culture media
prior to its use in the manufacturing process.

The studies presented here investigated the feasibility of implementing
culture media virus filtration with respect to its virus clearance
capacity under extreme conditions, which have been shown to be
worst-case for virus removal, such as high process feed loading, long
duration of filtration, and multiple process interruptions.

For the many experiments reported here, Minute virus of mice (MMV) was
chosen as small challenge virus. This is based on the fact that although
culture media increasingly do not contain animal derived components
(Grillberger~(2009)) viral contaminations of cell derived bulk harvests
have still occurred, e.g., with MMV (Garnick, (1998); Nims, (2006)).
Therefore, MMV is a target virus. Furthermore, MMV represents small
non-enveloped viruses which are the main challenge for virus filters
with a stipulated pore-size of 20 nm and is thus also a suitable model
virus (EMEA 1996).

\textbf{Materials and Methods}

\textbf{Culture Media}

For the small scale virus removal studies, including validation studies
according to guidance (EMEA, 1996), fermenter feed media used for the
manufacture of a biopharmaceutical product was used. The media consists
of DMEM / HAMS F12 to which Ethanolamine, Poloxamer 188, Soy Peptone,
L-Glutamine, NaHCO\textsubscript{3} and Copper were added. The pH of the
media was initially adjusted to 7.2 to 7.6. Soy-peptone (as
animal-derived material free nutrient) was added in different
concentrations and from different manufacturers.

In order to avoid bacterial contamination during the virus filtration
experiments, antibiotics (Gentamicin 0.1 g/L and optionally Penicillin
90 U/L with Streptomycin 0.1 g/L) were added to the un-spiked media.

\textbf{Equipment}

The virus removal filtration equipment consisted of an electronic
recorder, two pairs of redundant pressure sensors (placed up- and
down-stream of the pre-filter) and temperature sensors as generally
described elsewhere (Wieser, (2015)).

A pressure vessel made of electropolished 316L steel with a capacity of
5~Liters that could be pressurized to 7 bar (Millipore Cat. No.
XX6700P05) was used as a feed tank.

Pressure was applied to the tank via a manual controller for nitrogen
gas pressure. The nitrogen gas was passed over a 0.2\selectlanguage{greek}µ\selectlanguage{english}m air filter in
order to avoid introduction of bacterial contaminants.

The filtrate was collected in vessels placed on an electronic balance.
The data collected by electronic recorder was used to calculate process
feed loads, feed flow rates, process duration and duration of pressure
interruptions.

The virus removal filters used were:

\begin{itemize}
\tightlist
\item
  1\textsuperscript{st} generation filters:
\item
  Asahi Planova (Asahi Kasei Medical Co., Ltd., Tokyo, Japan) 15N
  filters; 10 cm\selectlanguage{ngerman}², rating: mean pore size 15~±~2~nm (Asahi KASEI,
  (2018))
\item
  Millipore (Merck KGaA, Darmstadt, Germany) NFP; 3.5 cm², rating:
  \textgreater{}4 log removal of small viruses (Merck Millipore, (2012))
\item
  Pall DV20 (Pall Corp., Port Washington, USA); 10 cm², rating:
  \textgreater{} 3 log for 20~nm viruses (Pall Life Sciences, (2010))
\item
  Pall Pegasus SV4; 9.6 cm², rating: 20 nm (nominal) (Pall Life
  Sciences, (2012))
\item
  2\textsuperscript{nd} generation filters
\item
  Asahi Planova BioEX filters; 3 cm², rating: designed to deliver
  \textgreater{} 4.0 logs of parvovirus removal (Asahi KASEI, (2018))
\item
  Millipore Barrier; 3.1 cm², rating: \selectlanguage{english}[?] 3.0 log removal of parvovirus
  (Merck Millipore, (2021))
\item
  Pall Pegasus Prime; 2.8 cm2, rating: 20 nm (nominal) (Pall Life
  Sciences, (2016))
\item
  Sartorius CPV; 5 cm2, rating: 20 nm (nominal) (Sartorius Stedim
  Biotech GmbH, (2011))
\item
  Sartorius HF; 1.7 cm2, rating: 20 nm (nominal) (Sartorius Stedim
  Biotech GmbH, (2014))
\end{itemize}

Each set-up included a Millipore Durapore pre-filter (rating: 0.1 \selectlanguage{greek}µ\selectlanguage{english}m
(nominal), 13 cm\selectlanguage{ngerman}²) set-up in-line in a dead-end filtration
configuration. The 0.1 \selectlanguage{greek}µ\selectlanguage{english}m pre-filter was flushed and conditioned with
water for injection before it was autoclaved at 100\selectlanguage{ngerman}°C for 30 minutes.
Then it was re-introduced into the filtration equipment and conditioned
with WFI again (together with the virus removal filter).

0.1\selectlanguage{greek}µ\selectlanguage{english}m Pall Acrodisc syringe filters were used to remove virus aggregates
from the thawed virus stocks.

The virus-spiked fermenter feed media was passed over a 0.2 \selectlanguage{greek}µ\selectlanguage{english}m Pall
Vacucap~90PF filter as needed in order to avoid the introduction of
bacterial contaminants.

The pre-use suitability of each viral filter used was verified according
to the vendors instructions:

Asahi filters were flushed with WFI, subjected to a pre-use air leakage
test and then filled with WFI again. Only filters passing this pre-use
test were used for individual experiments -- none of the filters tested
failed.

All other filters were subjected to normalized water flow tests during
which water was passed through the filter at specified pressures and the
flow rate measured. Only filters with water flow rates within the ranges
specified by the manufacturer were used for individual experiments --
none of the filters failed.

After each experiment the integrity of each viral filter used was
verified according to the vendors instructions. For the Asahi filters
this was identical to the pre-use leakage test. For all other filters
the test was a pressure hold test. Only filters for which no air was
seen exiting the filter at the specified testing pressure within a given
time period were seen to be integral.

\textbf{Viruses and virus assays}

MMV (ATCC VR-1346, strain Prototype; Family \emph{Parvoviridae} ;
non-enveloped ssDNA virus; \selectlanguage{ngerman}Ø~=~18~-~26~nm) was propagated and titrated
on A9 cells (ATCC CCL-1.4), as previously reported Berting, (2010).

Titers from the end-point dilution assays were expressed as
log\textsubscript{10} 50\% tissue culture infectious dose/mL
(TCID\textsubscript{50}/mL) and any influences of the process material
upon the virus infectivity assay characteristics, cell toxicity and
interference were considered.

Analysis of the virus removal capacity of the process was carried out as
described in or according to regulatory guidance (EMEA, (1996)). The
virus load before and after treatment were used to calculate the
logarithmic virus reduction factor (R). It is noted that the ``after
treatment'' virus load is the sum of the virus loads of each filtrate
fraction drawn during an experiment. Whenever virus was undetectable,
the limit of detection was taken as the virus titer for calculation.

\textbf{General Procedure}

The laboratory scale experiments were designed to simulate the feeding
of a perfusion fermenter with a fermenter feed media solution containing
MMV as a contaminant. The intention of each laboratory scale experiment
was to investigate the maximum process feed load capacity of an
investigated filter when loading it with fermenter feed media spiked
with MMV at a ratio of 1:250 (i.e., 0.4\% spike load). The MMV-spiked,
0.2 \selectlanguage{greek}µ\selectlanguage{english}m filtered fermenter feed media solution (``spiked starting
material'') was passed over a filter train consisting of one 0.1 \selectlanguage{greek}µ\selectlanguage{english}m
prefilter followed by a virus removal filter (each with a stipulated
pore size of approximately 20 nm, see Materials and Methods). During
each investigation the impact of multiple interruptions during the
loading process on virus clearance of a given filter was also
investigated. This was done when the filtrate collecting flask was
replaced by a fresh one (samples for virus titration were drawn from the
filtrate and stored at [?]-60\selectlanguage{ngerman}°C until the end of the experiment when all
filtrate samples were titrated together) -- during this activity
filtration was interrupted intentionally for at least one hour. One of
these intentional interruptions lasted at least 72~hours to represent
the pause between the first filling of the fermenter and the build-up of
the inoculum before the actual perfusion fermentation started. Filtrate
collection flask replacement -- and thus process interruption -- took
place approximately once a day. The overall number of interruptions per
experiment ranged from 1 to 21 depending on filter performance.
Filtration was continued until a flow decay of around 90\% was reached
or the duration of the experiment had exceeded 30 days (duration ranged
from one to 34 days). Virus removal filtration was performed at constant
transmembrane pressures (TMP, i.e., the pressure difference between feed
and filtrate -- which was at ambient pressure and, therefore, taken as
0.0 bar for calculations) over the virus removal filter. The TMP was set
to 1.0 to 2.6 bar for each experiment (the maximum TMP set-point was
determined either by the maximum value suggested by the filter
manufacturer or by the maximum pressure allowed by the tubing used) and
to 0.0 bar during the intentional interruptions. Filtration was
performed under ambient temperature for all experiments. pH was measured
for the spiked starting material and for each filtrate fraction.

Controls for aggregates (EMEA, (2010)) were performed for (a) the virus
stock and (b) the spiked starting material matrix. For (a) the titers of
the thawed virus stock and of the 0.1 \selectlanguage{greek}µ\selectlanguage{english}m filtered virus stock (which
then was used for spiking) were compared -- a titer reduction of less
than 1 log\textsubscript{10} is not considered to be significant
according to regulatory guidance (EMEA, (1996)). For (b) the titer of
the spiked starting material was compared to the titer of the
pre-filtrate drawn during the first interruption.

In order to evaluate the influence of the freeze/thaw cycle, to which
the filtrate fractions were subjected, on the virus titer of these
samples, freeze controls were performed (EMEA, (1996)). Two freeze
control levels were investigated: (c) ``high titer'' and (d) ``low
titer''. For (c) the titer of the spiked starting material (titrated
immediately) was compared to the titer of the same matrix which had been
stored at [?]-60\selectlanguage{ngerman}°C throughout the experiment and titrated together with
all other samples which had been stored frozen. For (d) the titer of the
spiked starting material diluted with starting material to a theoretical
MMV titer of 2.0 log\textsubscript{10} {[}TCID\textsubscript{50}/mL{]}
(titrated immediately) was compared to the titer of the same matrix
which had been stored at \selectlanguage{english}[?]-60degC throughout the experiment and titrated
together with all other samples which had been stored frozen.

In order to evaluate the influence of ambient conditions during an
experiment on the virus titer, process controls were performed. To this
purpose an aliquot of the spiked starting material was kept at ambient
temperature in the biosafety cabinet where the virus filtration was
performed, until the end of the NF procedure. At evenly spaced intervals
samples were drawn and stored at \selectlanguage{english}[?]-60degC throughout the experiment and
titrated together with all other samples which had been stored frozen.
The virus titers of these samples were compared to that of the spiked
starting material (titrated immediately).

Un-spiked control runs were performed for the virus clearance validation
studies. All settings were identical to those for the virus spiked runs
with the exception that no intentional pressure interruptions were
performed. Samples were drawn for the analysis of glucose concentration
and osmolality. Furthermore, a cell proliferation use-test was performed
with the pooled filtrate in order to evaluate if the composition of the
fermenter feed media was significantly altered during filtration with
respect to supporting cell growth. The use test consisted of the
analysis of the cell density, cell viability and cell productivity after
a 3-day incubation period with the filtered media.



\textbf{Results}

The aim of this investigation was to evaluate which virus filters were
suitable for culture media virus filtration. This was done by
investigating the MMV removal capacity of different virus filters under
the conditions which can be expected when filtering culture media. The
intention of each laboratory scale experiment was to investigate the
maximum process feed load capacity of an investigated filter when
loading it with MMV-spiked fermenter feed media whilst demonstrating an
effective virus clearance capacity with no to minimal virus
breakthrough.

The culture media used for all experiments contained a base media with
an anti-foaming agent and a wide variety of nutrients typical for high
performance process. Soy-peptone (as animal-derived component free
nutrient) was added in different concentrations and from different
manufacturers -- these variations were, however, not seen to have any
impact on the virus clearance capacity of the investigated filters and
are, therefore, not discussed in further detail here.

MMV was chosen as -- on the one side -- it is a target virus as a
contaminant for some cell cultures -- such as CHO (Barone, (2020)), and
-- on the other hand -- it represents small non-enveloped viruses which
are the main challenge for the investigated virus filters with a
stipulated pore-size of about 20~nm.

The worst-case conditions investigated in this study were transmembrane
pressure (TMP) -- including pressure interruptions, duration of
filtration, and process feed load (PDA, (2022)).

The TMP set-point (which is reflected in the mean TMP measured during
the filtration phases) was determined either by the maximum value
suggested by the filter manufacturer or by the maximum pressure allowed
by the tubing used (i.e., 2.6 bar). The complete TMP range was bracketed
by intentionally introducing multiple process interruptions. These were
typically one \selectlanguage{english}[?]~72~hour pause (representing e.g., the duration of
inoculum build-up in the fermenter), followed by daily \selectlanguage{english}[?]~1~hour pauses.
During some experiments a lower mean TMP (i.e., 1 bar) was also
investigated in order to broaden the data base.

Duration was a decision point, as the goal was to continue filtration
for at least 30 days or until flow decay reached or exceeded 90\%
(sometimes earlier if a decision had to be made whether the feed tank
should be refilled). As a result of these decision points the maximum
load and duration possible whilst maintaining effective virus clearance
could be determined.

The filters investigated are grouped into first- and second-generation
filters (Miesegaes, (2010)). First generation filters are designed for
down-stream manufacturing processes. Typically, these processes need
virus filters designed to handle up to 1,000 L/m2 of an aqueous protein
solution as rapidly as possible. Furthermore, first-generation filters
were not typically designed to tolerate the impact of pressure
interruptions or low flow phases on their virus retention capacities.
Second generation filters do generally have improved virus retention
capabilities even if pressure interruptions occur.

Table 1. Filtration conditions and results for MMV retention using first
generation (FG) filters.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Filter & FG I & FG II & FG III & FG IV\tabularnewline
\midrule
\endhead
Mean TMP {[}bar{]} & 2.1 & 2.1 & 2.1 & 1.1\tabularnewline
Process pauses & 10 * & 7 & 9 * & 6*\tabularnewline
flow decay {[}\%{]} & \textgreater{} 90 & \textgreater{} 90 &
\textgreater{} 90 & \textasciitilde{} 80\%\tabularnewline
Duration {[}days{]} & 11 & 14 & 14 & 7\tabularnewline
Media feed load {[}L/m\selectlanguage{ngerman}²{]} & \textbf{4,611} & \textbf{4,913} &
\textbf{4,511} & \textbf{4,841}\tabularnewline
Virus reduction factor (R) {[}log\textsubscript{10}{]} & \textbf{1.0} &
\textbf{1.1} & \textbf{2.6} & \textbf{4.4}\tabularnewline
Virus breakthrough? & yes & yes & yes & yes\tabularnewline
\bottomrule
\end{longtable}

* Short pauses (under 1 hour each)

Table 1 shows the results for the investigated first generation filters.
For all filters the decision point flow decay was reached rather than
duration. The MMV reduction factors (R) for the filters FG~I to FG~III
indicate significant virus breakthrough. These filters were, therefore,
unsuitable for high-volume long duration applications. Only filter FG~IV
showed effective virus clearance (4.4~log\textsubscript{10}) at a higher
than 1,000 L/m\selectlanguage{ngerman}² load. However, the experimental run was stopped after
6~days as the flow decay had already reached 80\%. The short duration of
the experiment indicated that the filter is not suitable for a
high-volume long duration application.

Table 2. Filtration conditions and results for MMV retention --
2\textsuperscript{nd} generation filter I (SG~I) -- Sartorius Sartopore
CPV.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
Mean TMP {[}bar{]} & 1.0 & 1.0 & 2.2 & 2.3 & 2.6\tabularnewline
\midrule
\endhead
Process pauses & 20 & 17 & 16 & 21 & 19\tabularnewline
flow decay {[}\%{]} & 78 & 90 & 76 & 80 & 78\tabularnewline
Duration {[}days{]} & 30 & 32 & 23 & 33 & 26\tabularnewline
Media feed load {[}L/m\selectlanguage{ngerman}²{]} & \textbf{10,242} & \textbf{9,273} &
\textbf{19,617} & \textbf{19,531} & \textbf{19,440}\tabularnewline
R {[}log\textsubscript{10}{]} & \textbf{\textgreater{} 6.1} &
\textbf{5.0} & \textbf{\textgreater{} 6.3} & \textbf{\textgreater{} 6.2}
& \textbf{\textgreater{} 6.5}\tabularnewline
Virus breakthrough? & no & minor & no & minimal & no\tabularnewline
\bottomrule
\end{longtable}

Per TMP set-point a maximum of two representative experiments are
presented here.

A ``\textgreater{}'' preceding the logarithmic reduction factor (R)
combined with a ``no'' result for the parameter ``virus breakthrough''
indicates removal below the limit of detection for all of the filtrate
fractions.

If minimal virus infectivity was found in a minority of the filtrate
fractions (i.e., the titers were at the limit of detection) then the
result for virus-breakthrough is ``minimal''.

A ``minor'' virus breakthrough indicates that minimal infectivity was
found in the majority of the filtrate fractions.

Table 3. Filtration conditions and results for MMV retention --
2\textsuperscript{nd} generation filter II (SG~II) -- ASAHI Planova
BioEX.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Mean TMP {[}bar{]} & 1.0 & 1.0 & 2.4 & 2.4\tabularnewline
\midrule
\endhead
Process pauses & 18 & 19 & 14 & 14\tabularnewline
flow decay {[}\%{]} & 87 & \textgreater{} 90 & \textgreater{} 90 &
\textgreater{} 90\tabularnewline
Duration {[}days{]} & 30 & 34 & 27 & 27\tabularnewline
Media feed load {[}L/m\selectlanguage{ngerman}²{]} & \textbf{9,043} & \textbf{7,845} &
\textbf{15,504} & \textbf{14,098}\tabularnewline
R {[}log\textsubscript{10}{]} & \textbf{\textgreater{} 6.5} &
\textbf{\textgreater{} 5.6} & \textbf{\textgreater{} 6.1} &
\textbf{\textgreater{} 5.8}\tabularnewline
Virus breakthrough? & no & minimal & minimal & minimal\tabularnewline
\bottomrule
\end{longtable}

A ``\textgreater{}'' preceding the logarithmic reduction factor (R)
combined with a ``no'' result for the parameter ``virus breakthrough''
indicates removal below the limit of detection for all of the filtrate
fractions.

If minimal virus infectivity was found in a minority of the filtrate
fractions (i.e., the titers were at the limit of detection) then the
result for virus-breakthrough is ``minimal''.

The early 2\textsuperscript{nd} generation down-stream filters
investigated (filters SG~I and SG~II -- see tables 2 and 3), however,
were found to have robust virus reduction capacities even under worst
case load and duration conditions. Virus reduction was found to be
effective for both filters (R ranging from 5.0~log\textsubscript{10} to
\textgreater{}~6.5 log\textsubscript{10}) although some minimal virus
break-through was found in several fractions. Variation of the TMP had
no impact on the virus clearance capacity of either of the filters --
although it did impact the process feed loads possible before reaching
any of the decision points.

Both filters are deemed to be equally suitable for the high-volume long
duration filtration which would be needed for a perfusion reactor -- and
thus also for any smaller volume set-up, including batch filtrations.

It is noted that biochemical parameters, i.e., glucose concentration and
osmolality pre and post filtration, confirmed that virus filtration had
no significant impact on the composition of the fermenter feed media.
Furthermore, the use test regarding cell culture performance with the
filtered media showed cell density, cell viability and cell productivity
(rate of expression of the target protein) to be within specifications,
and fully equivalent to that of unfiltered media (data not shown).

Table 4. Filtration conditions and results for MMV retention using
improved second generation filters SG III to SG VI.\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
Filter & SG III & SG III & SG IV & SG IV & SG V & SG V & SG VI & SG
VI\tabularnewline
\midrule
\endhead
Mean TMP {[}bar{]} & 2.0 & 2.1 & 2.1 & 2.1 & 2.1 & 2.1 & 2.2 &
2.1\tabularnewline
Process pauses & 3 & 4 & 2 & 1 & 4 & 2 & 8 & 8\tabularnewline
flow decay {[}\%{]} & \textgreater{} 90 & \textgreater{} 90 &
\textgreater{} 90 & \textgreater{} 90 & 90\% & \textgreater{} 90 &
\textgreater{} 90 & \textgreater{} 90\tabularnewline
Duration {[}days{]} & 6 & 6 & 2 & 1 & 9 & 6 & 13 & 13\tabularnewline
Media feed load {[}L/m\selectlanguage{ngerman}²{]} & \textbf{10,500} & \textbf{8,700} &
\textbf{3,893} & \textbf{3,582} & \textbf{15,700} & \textbf{11,400} &
\textbf{11,500} & \textbf{11,000}\tabularnewline
R {[}log\textsubscript{10}{]} & \textbf{\textgreater{} 5.3} &
\textbf{\textgreater{} 5.8} & \textbf{4.4} & \textbf{3.7} & \textbf{4.3}
& \textbf{4.4} & \textbf{\textgreater{} 6.3} & \textbf{\textgreater{}
6.2}\tabularnewline
Virus break-through? & no & no & yes & yes & yes & yes & minimal &
minimal\tabularnewline
\bottomrule
\end{longtable}

A ``\textgreater{}'' preceding the logarithmic reduction factor (R)
combined with a ``no'' result for the parameter ``virus breakthrough''
indicates removal below the limit of detection for all of the filtrate
fractions.

If minimal virus infectivity was found in a minority of the filtrate
fractions (i.e., the titers were at the limit of detection) then the
result for virus-breakthrough is ``minimal''.

In the meanwhile, new filters are continuously being developed -- some
of which focus on increasing throughput whilst maintaining virus
clearance capacity. These improved 2\textsuperscript{nd} generation
filters reduce the filter area demand for large volume virus filtration,
and thus reduce facility floor footprint for large-volume filtration, as
well as filter cost.

All investigated filters showed a significantly higher initial feed flow
rate than the until then commercially available filters (see Table 4).
This led to higher initial throughput, but filter fouling occurred
relatively early for all filters.

For filter SG~IV this is probably the reason for the termination of the
process due to significant flow decay within 1 to 2 days after up to
3,900 L/m\selectlanguage{ngerman}² had been filtered. For this filter virus reduction was
effective -- with mean R values of 4.4 and 3.7~log\textsubscript{10} but
virus breakthrough was observed in most filtrate fractions. Nonetheless,
this filter is a significant improvement to filter FG~III from the same
manufacturer, with which only 2.6 log\textsubscript{10} MMV reduction
was achieved. This filter could be suitable for batch filtration prior
to the actual fermenter process.

Filter SG~V showed an increased initial flow rate when compared to
filter SG~I of the same manufacturer, but all other parameters such as
throughput and duration were not as good. Furthermore, although virus
reduction was effective (mean R value: 4.4~log\textsubscript{10}) it was
significantly lower than what was achieved for filter SG~I (5.0 to
\textgreater{}6.5~log\textsubscript{10}).

Filter SG~III shows a significant improvement of the filter
characteristics with respect to high volume long duration filtration
when compared to filters FG~I and FG~II from the same vendor. The same
can be said about developmental filter SG~VI when compared to filter
FG~IV of the same manufacturer (however, when compared to filter SG~II
-- which is an early 2\textsuperscript{nd} generation filter from the
same vendor -- no significant improvements can be seen).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure 1. Overview of the development of the logarithmic reduction
factor (R) during representative runs for each filter investigated.
Arrows pointing up indicate the corresponding filtrate fraction titer
was below the limit of detection.

The virus reduction factor values (R) in relation to filtered volumes
are summarized in Figure 1. Filters FG~I to FG~III exhibited significant
MMV breakthrough resulting in a rapidly decreasing R values -- even
though initial values (when volumes were near to the volumes the filters
were designed for) were not unpromising. Filter FG~IV shows effective
virus clearance, however, with some significant virus breakthrough after
a loading of more than 2000 L/m\selectlanguage{ngerman}² or due to process interruptions. All
other filters show effective virus clearance, whereby filters SG~I and
SG~II show the best virus clearance capacities at the highest process
feed loads and were thus best suited for the intended purpose.

\textbf{Discussion}

Raw materials or cell culture media including specific components
thereof, like FBS, have been identified as source of viral contaminants
for biopharmaceutical products based on eucaryotic expression systems.
It has been reported that such contaminations have led to virus
contamination and the subsequent collapse of the cell population in
these fermenters. As a result, severe measures to clean up the upstream,
and should any intermediate have been forward-processed already also the
down-stream equipment, to minimize the recurrence of such an event had
to be taken. The downtime can lead to unmet patient needs, dramatic
economic consequences, and severe impact on the reputation of an
affected company (Bethencourt, (2009)).

The introduction of a contaminating virus might not necessarily become
obvious by degenerative effects presented by the production cells, as
``silent infections'' have also occurred (Moody, (2011)). The specific
adventitious agent testing (AAT) program on fermenter harvest material
would be the point where a virus contaminant would be detected. However
not necessarily: although AAT is designed to detect as many potential
viral contaminants as possible, there might be viruses that would not be
picked up (Gombold, (2014)). Similarly, the testing of raw materials
does not necessarily reveal a viral contaminant, especially when the
concentration of the contaminant in the raw material is lower than the
detection limit of the testing procedure, or the contaminant is unevenly
distributed across the large volumes involved versus the small volumes
actually tested.

Therefore, to make sure that a potential contaminant would never reach
the patient, a series of effective virus clearance steps is implemented
in down-stream biomanufacturing operations, where feasible. Some
biopharmaceutical products can, however, not be subjected to dedicated
viral clearance steps in the down-stream processes as they would not
only inactivate or remove potentially present viral contaminations but
have the same clearance effect on the biopharmaceutical components. This
is true for live virus vaccines, ATMPs (e.g., cellular or gene
therapies) or particularly complex biotechnology products which are
simply too large to be subjected to filtration with virus filters with a
nominal pore size of 20~nm, e.g.: vWF (Parker, (2021)).

A potential solution is to introduce a method which ensures a
significant reduction of theoretically present viruses in the culture
media before it even enters a manufacturing process, i.e., an upstream
virus barrier.

Different methods capable of this feat have been investigated: high
temperature short time treatment (HTST), ultraviolet light irradiation
(UV-C) and finally virus filtration. The implementation of HTST and UV-C
have, however, been faced with complications as in both cases media
components are susceptible to degeneration (Cao, (2013)), (Meunier,
(2016)).

Virus filtration, however, is one of the most robust and effective virus
clearance procedures, as it conceptually removes all pathogens as long
as their size is larger than the stipulated pore-size of the filter,
while at the same time being neutral to the biological activity of the
biopharmaceutical compound(s) smaller than the pore size. It is,
therefore, best suited to reduce the risk of the introduction of
adventitious viruses through culture media. For this process to be
economically feasible the filter area must ideally be kept small while
at the same time large volumes of culture media are processed for a
potentially prolonged times of filtration, and the impact of low or no
flow incidents on the virus clearance capacity needs to be understood
also (Wieser, (2016)).

The studies presented here investigated the feasibility of implementing
culture media virus filtration with respect to their virus clearance
capacities under extreme conditions such as very high process feed
loading (up to \textasciitilde{} 19,000 L/m²), very long duration (up to
31 days), and multiple process interruptions (up to 21, with cumulative
interruptions of over 92 hours). MMV was used as a relevant target virus
as a contaminant for some cell cultures, and in general as a small
non-enveloped viruses which are the main challenge for the investigated
virus filters with a stipulated pore-size of about 20~nm.

It was found that a number of filters -- especially of the newer
2\textsuperscript{nd} generation -- are capable of effective virus
clearance despite the harsh regimen they were subjected to. At the same
time the investigation of biochemical parameters for un-spiked control
runs showed the filters to have no measurable impact on the composition
of the culture media. Again, indicting this technology to be especially
suitable for large volume pre-manufacturing process culture media
preparation.

\textbf{Acknowledgements}

The authors contributed equally to this manuscript. The contributions of
the entire Takeda Global Pathogen Safety team, most notably Brigitte
Kainz and Michaela Rumpold (conduct of experiments), are herewith
gratefully acknowledged.

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https://doi.org/10.5731/pdajpst.2015.006346\selectlanguage{english}
\begin{figure}[H]
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