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\begin{document}

\title{Novel spiking methods developed for anion exchange chromatography
operating in continuous processing}



\author[1]{Ying Li}%
\author[2]{Audrey Chang}%
\author[1]{David Beattie}%
\author[3]{Kathryn Remington}%
\affil[1]{MilliporeSigma Process Solutions Research and Development}%
\affil[2]{MilliporeSigma}%
\affil[3]{MilliporeSigma Process Solutions Services }%


\vspace{-1em}



  
  \date{May 31, 2024}


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\selectlanguage{english}
\begin{abstract}
Many manufacturers of biopharmaceuticals are moving from batch to
continuous processing. While this approach offers advantages over batch
processing to manufacturers, demonstration of viral clearance for
continuous processes is more complex. Regulators expect manufacturers to
use an appropriate scale down model, based on solid scientific
justification, to verify the viral reduction capacity of the
manufacturing process. The output from chromatography columns operated
in continuous processes fluctuates in concentration so that the load for
the subsequent column is not homogenous. This must be considered when
designing viral clearance studies. One way to approach clearance studies
is to downscale the continuous process, using multi-column
chromatography systems and in-line spiking of virus. The multi-column
chromatography systems and experienced operators, however, may not be
available at the CRO performing the study. Another approach is to
evaluate each step in traditional batch mode, using existing
chromatography systems, but to modify the spiking and loading conditions
to mimic the variance introduced by the transition between the two
connected process steps. Using a standard chromatography system, we have
evaluated a flow through anion exchange chromatography step in a
monoclonal antibody process using five different methods to introduce
the virus to the column. We have shown that regardless of whether the
virus spike is introduced in a well-mixed batch mode, introduced as a
concentrated pulse of virus with homogeneous mAb or with a concentrated
peak of mAb, the clearance of MMV was similar. This study introduces an
alternative way to evaluate viral clearance in a continuous process.%
\end{abstract}%



\sloppy


\section*{1. Introduction}

{\label{550371}}

There is a current trend in biomanufacturing to move from batch to
continuous processing (Bisschops \& Brower, 2013; Godawat et al., 2015;
Konstantinov \& Cooney, 2015). This shift offers a number of potential
advantages, such as greater efficiency, improved product quality,
decreased production costs and a smaller equipment footprint. It also
increases the flexibility for a manufacturer to produce both large and
small volume biopharmaceuticals and to adjust production capacity to
demands (Croughan et al., 2015; Zydney, 2015). Manufacturers can produce
more easily stable proteins as well as labile enzymes and clotting
factors that cannot tolerate the bulk harvest hold times often
associated with batch manufacturing. Perfusion bioreactors equipped with
cell retention devices have been used to create a continuous upstream
process. Increased expression levels of recombinant proteins in a
continuous upstream create a bottleneck between the upstream and
downstream processes (Jungbauer, 2013). Capture columns, such as a
Protein A column for monoclonal antibodies, can be increased in size or
cycled multiple times during a batch to mitigate the bottleneck;
however, for many reasons, these approaches are unsatisfactory
(Bisschops \& Brower, 2013). Continuous processing using multi-column
chromatography can successfully integrate the upstream process with the
downstream process and ultimately lead to steady-state manufacturing
(Bisschops \& Brower, 2013; Ichihara et al., 2018; Konstantinov \&
Cooney, 2015). A number of chromatographic systems have been developed
for continuous product capture including the periodic counter-current
chromatography (PCC) and simulated moving bed chromatography (SMB)
systems (Zydney, 2016; Bryntesson et al., 2011; Godawat et al., 2012).
Chromatographic unit operations run in flow-through mode are often used
as polishing steps in the purification of biologics and adapt well to a
continuous process (Weaver et al., 2013; Ichihara et al., 2018).
Operation in flow-through mode usually enables higher column loading and
results in a more robust unit operation (Shukla et al., 2007). Anion
exchange chromatography (AEX), is often used as a combined intermediate
and polishing step in the purification of monoclonal antibodies (mAb)
and can operate in flow-through mode using either resin-packed-bed
columns or membrane adsorbers (Zhou, 2006). When used this way, the
positively charged matrix binds negatively charged impurities, such as
host cell proteins and DNA, and allows the product molecule to flow
through the chromatography matrix unimpeded. Under the appropriate
conductivity and pH conditions, this step can also provide good viral
clearance, if there is a difference in isoelectric point (pI) between
virus and the protein product (Miesegaes, 2014). Like manufacturing
processes run in batch mode, ensuring the viral safety of products
manufactured from continuous bioprocesses is critical to the overall
safety of the product. Screening of raw materials and use of virus
barrier methods for culture medium will continue to be key viral safety
strategies (ICH, 1999; EMA, 1996) and important for continuous
processes. The viral safety of continuous processes ultimately may
involve in-line, on-line or at-line process analytical technology (PAT)
methods because the typical assays for detection of adventitious
infectious agents require too much time to be used in a continuous
process (Holzer, 2018). New approaches to the evaluation of the viral
reduction capacity of the manufacturing process may be needed, including
advanced molecular methods (Johnson et al., 2017). With batch mode
manufacturing, individual processing steps can be scaled down
independently, a virus spike introduced into the load material and then
the step assessed for its ability to inactivate or remove virus (EMA,
1996). A continuous process, however, introduces complications into the
design of an appropriate scale down model and requires consideration of
the linked unit operations. Regulatory authorities understand the
advantages of the continuous processing approach to the manufacture of
biopharmaceuticals however, the FDA have clearly indicated the
expectation for manufacturers to use an appropriate model based on solid
scientific justification to verify the viral clearance capacity of the
manufacturing process (Johnson et al., 2017). Evaluation of viral
clearance for linked unit operations in continuous processing is complex
and manufacturers are beginning to explore the best way to implement it.
One approach to this is to develop a valid lab scale model for an
integrated continuous process employing multiple unit operations, each
of which provides a significant level of virus removal. Virus is
introduced by in-line spiking and sampling is performed using the method
previously established by Lutz, et al. (2011). This may provide a
representative model for a physically connected process. However
challenges exist in adoption of this approach for regulated viral
clearance studies. First, it is difficult to set up and evaluate such
connected processes in a contract viral clearance testing laboratory
given the time and level of expertise required. Each manufacturer may
use different methods and equipment for continuous processing which
introduces additional complexity. Secondly, virus needs to be spiked at
multiple unit operations to provide enough virus to determine the viral
reduction potential of the entire process. This creates additional
complexity in the design of experiments as well as the collection and
analysis of samples. Finally, in a typical integrated continuous
process, the feed to a particular unit operation may come directly from
a previous unit operation, instead of a well-mixed storage tank as in
batch processing. While column feeds coming from a column immediately
upstream will fluctuate in concentration, the virus spike introduced
with in-line spiking would be at a constant concentration, and it is
unclear whether this represents the way a potential contaminating virus
would enter the column. Another approach is to validate each unit
operation in batch mode, but to modify the spiking and loading
conditions to mimic the variance introduced by the transition between
two connected unit operations. The advantage of this approach is that it
can be readily implemented in a viral clearance testing facility using
existing chromatography systems. However, solid scientific proof is
required to demonstrate that the viral clearance study is representative
of continuous processing. A comprehensive study covering the entire
design space will be needed to address the potential risks introduced by
continuous processing; this may involve many experiments following
design of experiment (DOE) principles (Strauss et al., 2009a; Strauss et
al., 2010). Angelo et al. (2019) developed a surrogate model using
standard batch mode chromatography to mimic the operation of multicolumn
capture chromatography and demonstrated that the viral clearance
performance was comparable between the batch model and a two-column
continuous system. To evaluate this, we used a standard chromatography
system to evaluate viral clearance across the flow through AEX step of a
monoclonal antibody process, using various methods to introduce virus to
the column. These methods may be useful when assessing the viral
clearance capacity of an AEX column operated in a connected process.

\section*{2. Materials and Methods}

{\label{430369}}

\subsection*{2.1 Feed stocks}

{\label{781130}}

Feed material was a monoclonal antibody (IgG1) generated internally by
MilliporeSigma (Bedford, MA). The harvested cell culture was purified by
Clarisolve\selectlanguage{ngerman}\textsuperscript{®} Pod (MilliporeSigma/Merck KGaA, Darmstadt,
Germany) clarification, Eshmuno\textsuperscript{®}A
(MilliporeSigma/Merck KGaA, Darmstadt, Germany) affinity chromatography
and stored at -80°C. The conductivity and pH of Protein A eluate were
adjusted to 6.0 ± 0.5 mS/cm and 8.5 ± 0.1 in 25mM Tris-HCl buffer by
adding 5 M sodium chloride (NaCl) and 2M Tris respectively. The
feedstock was filtered through a 0.22 \selectlanguage{greek}µ\selectlanguage{english}m filter prior to use.

\subsection*{2.2 Virus Stocks and Assays}

{\label{643191}}

Mouse minute virus (MMV) stocks were provided by MilliporeSigma
(Rockville, MD). A single lot of MMV with a certified titer of 8.6 \selectlanguage{ngerman}± 0.3
log\textsubscript{10} TCID\textsubscript{50}/mL was used for all runs.
The MMV titer for each test sample was quantified by 50\% tissue culture
infectious dose (TCID\textsubscript{50}) endpoint assay using 324K cells
and calculated using Spearman Kärber method (Hamilton et al., 1977;
Schmidt et al., 1989), according to MilliporeSigma standard operating
procedures. Preliminary cytotoxicity and viral interference testing were
performed before the study, and a minimum five-fold pre-dilution was
necessary to mitigate any cytotoxicity and viral interference within the
infectivity assay systems. Log\textsubscript{10} reduction values (LRVs)
were calculated as the logarithmic reduction of total virus particles as
recommended by the International Conference on Harmonization Q5A.

The mAb concentrations were assessed by UV-Vis
A\textsubscript{280}absorbance, using an extinction coefficient of 1.47.
Residual CHO host cell proteins (CHO-HCP) were measured by CHO ELISA
kits (Cygnus Technologies, Southport, NC).

\subsection*{2.3 Anion exchange
chromatography}

{\label{403357}}

Chromatography columns (0.5cm diameter, 20cm bed height, 4mL column
volume, OPUS\textsuperscript{®}~ValiChrom\selectlanguage{english}) prepacked with
Eshmuno\textsuperscript{(r)} A (MilliporeSigma/Merck KGaA, Darmstadt,
Germany) resin in 20\% ethanol/150 mM NaCl were purchased from Repligen
(Waltham, MA). Chromatography runs were conducted on an AKTA Pure25 (GE
Healthcare, Chicago, IL). The total mAb load was maintained at 175 +-
10mg/mL for all runs, and the flow rate was set at 1mL/min to achieve a
contact time with the resin of 4min. The column was first equilibrated
with five column volumes (CVs) of equilibration buffer (Tris-HCl at 6.0
+- 0.5 mS/cm conductivity and 8.5 +- 0.1 pH). The mAb feed pools were then
loaded onto the column with different spiking/loading patterns as
described in Section 2.4, and four flow-through fractions of five CV
each were collected. At the end of the load phase, the column was washed
with five CV of equilibration buffer and the collection of this wash
fraction was terminated when OD\textsubscript{280} dropped to less than
50mV. The wash was collected and assayed as a fifth fraction, and a
percentage of each of the five fractions was pooled and assayed for
infectious virus and CHO-HCP. The column was stripped with 25mM Tris
buffer with 1M NaCl, sanitized with 1M sodium hydroxide (NaOH) and
stored in 20\% ethanol with 150mM NaCl after each run.

\subsection*{2.4 Viral spiking and loading
methods}

{\label{433943}}

Five different methods for introduction of the virus spike and the mAb
column feed to the AEX column were evaluated (Figure 1). For all
methods, 80 mL of mAb feed was loaded onto the column along with a
targeted total MMV load of 8.0
log\textsubscript{10}TCID\textsubscript{50}.\emph{Method 1} represents
the virus-spiked loading for a downscaled model of a batch process,
typically used in a viral clearance study, in which a homogeneous
mixture of virus spike and mAb feed are loaded onto the column. To
prepare the spiked load, 100 mL of 8.8 +- 0.5 g/L mAb feed was spiked
with 0.33mL MMV stock, and the resultant MMV concentration was measured
to be 6.3 +- 0.3 log\textsubscript{10}TCID\textsubscript{50}/mL (a total
of 8.2 log\textsubscript{10}TCID\textsubscript{50} MMV). A total of 20CV
(80mL) of the spiked mAb feed was loaded onto the column during the load
phase. Methods 2 and 3 were designed to explore the binding of virus on
AEX resins while loading a constant concentration of mAb.\emph{Method 2}
was designed to introduce a pulse of virus. To do this, the mAb feed was
split into two pools (18CV of pool A, 2CV of pool B). MMV stock (0.33mL)
was spiked into pool B only. The total MMV spiked into this small pool
was 7.8 +- 0.3 log\textsubscript{10}TCID\textsubscript{50}. The load
phase, 9CV of pool A was loaded onto the column, followed by 2CV of the
spiked pool B, and finally, the remainder of unspiked pool A. This
method introduced a bolus of MMV, at a concentration of 6.9 +- 0.3
log\textsubscript{10}TCID\textsubscript{50}/mL, mid-way through the
loading of the mAb.\emph{Method 3} was designed to represent the
introduction of a sharp pulse of virus. MMV stock (0.33mL) was mixed
with mAb feed to a total volume of 1mL and the mixture was loaded into
the 1mL sample loop. The total MMV loaded onto the column in this small
volume was measured to be 7.9 +- 0.3 log\textsubscript{10}
TCID\textsubscript{50}; the concentration was 7.9 +- 0.3
log\textsubscript{10}TCID\textsubscript{50}/mL. During the loading
phase, unspiked mAb feed was loaded onto the column for the first 10 CV,
as done with Method 1, the batch method. The remainder of the feed
solution was adjusted to flow through the 1mL sample loop, behind the
virus spike, before continuing to the column, resulting in the addition
of the virus spike in a short pulse of high titer virus.\emph{Method 4}
was designed to simulate fluctuating conditions that might be introduced
from connected, continuous processes. To simulate loading of feed
materials from elution of an upstream bind-and-elute chromatography
step, the mAb feed was split into two pools at different mAb
concentrations (18CV of pool A at a mAb concentration of 3.1 +- 0.1 g/L
and 2CV of pool B at a mAb concentration of 56 +- 2g/L). The high and low
concentration mAb solutions were prepared by dilution of the mAb stock
with process buffer. pH and conductivity were adjusted as described in
Section 2.1. MMV stock was spiked into pool B to a measured total 8.1 +-
0.3 log\textsubscript{10}TCID\textsubscript{50}; the concentration of
virus was 7.2 +- 0.3 log\textsubscript{10} TCID\textsubscript{50}/mL. The
load phase followed the same program as in Method 2, loading 9 CV of
pool A followed by 2 CV of the spiked pool B and then 9 CV of unspiked
pool A. This resulted in a peak of more highly concentrated mAb that
coincided with the pulse of MMV.\emph{Method 5} used in-line spiking to
deliver a constant concentration of virus spike, while the concentration
of mAb was varied. Pools of mAb feed were prepared as described in
Method 4; mAb concentrations of the larger pool was measured to be 3.0 +-
0.2g/L and the smaller pool to be 56 +- 2g/L. Instead of spiking virus
into a mAb pool, 0.33mL (measured 7.9 +- 0.3 log\textsubscript{10} total
TCID\textsubscript{50}) of virus stock was diluted with equilibration
buffer to a final volume of 4mL and loaded separately through system
pump B at a flow rate of 0.05mL/min. The virus stream and the mAb feed
stream were mixed in-line using the mixer in the AKTA system before
loading to the AEX column. The unspiked mAb pools were loaded using the
strategy of 9 CV of dilute mAb, then 2 CV of concentrated mAb, followed
by the final 9 CV of dilute mAb. The different spiking and loading
parameters for all 5 methods are summarized in Table 1. Duplicate
experiments were performed for each experimental condition with two AEX
columns.

\section*{3. Results and discussion}

{\label{689982}}

\subsection*{3.1 Viral and HCP clearance by different load and spike
methods}

{\label{156821}}

In this study, alternative spiking methods were evaluated for a
flow-through AEX unit operation run on a standard chromatography system.
AEX was selected as the test unit operation because it is a
chromatography unit operation that can provide significant virus removal
for many mAb processes. It is easier to detect variation in LRV
introduced by the modification of spiking methods with this step
compared with other chromatography modes such as Protein A affinity or
cation exchange chromatography (CEX) which only provide moderate to low
LRV. Nevertheless, the spiking methods described in this study can be
applied to other chromatography modes and operating conditions. The
different loading patterns described in Section 2.4 can be conveniently
programmed and then implemented on a standard chromatography system. AEX
removes virus primarily by electrostatic interactions between negatively
charged viruses and the positively charged ligand. Eshmuno Q is a strong
anion exchanger with trimethylammoniumethyl (TMAE) as the functional
group and is stable operating from pH 2 to 12. The viral clearance
performance of AEX is influenced by many variables, including the pH and
conductivity of the feed and process buffer solutions, load density,
flow rate and residence time, all of which have been widely explored in
many studies (Miesegaes et al., 2014; Roush, 2014; Strauss et al.,
2009a; Strauss et al, 2009b; Strauss et al., 2010). Eshmuno Q has been
shown to achieve good levels of viral clearance under the pH,
conductivity and residence time conditions used in this study, and this
was confirmed using spiking/loading Method 1 (Elich et al., 2019). The
current study focused on the impact of potential fluctuations in load
and viral spike concentrations that might be introduced by connected,
continuous processes, on the viral clearance performance of AEX in flow
through mode. Figure 1 is a schematic of the five different spiking and
loading methods developed to simulate potential scenarios in continuous
processing. For each method, the total virus remaining in each of the
collected flow through fractions, after being spiked with approximately
8.0 log\textsubscript{10} TCID\textsubscript{50} is shown in Figure 2.
The flow through was collected in four fractions of approximately equal
volume; the column wash was also collected as a separate, fifth
fraction. A small amount of virus is more readily detected in a smaller
volume than in a large volume and so by collecting the flow through in
fractions, unbound virus might be more readily detected. The LRVs for
runs using the different load and spike methods are summarized in Table
2. Method 1 is based on the conventional spiking design for viral
clearance evaluations of batch processes. In this method, the virus was
spiked into a well-mixed mAb feed pool; and the concentrations of mAb
and virus flowing through the column were constant throughout the
experiment. Except for the first flow-through fraction in which a small
amount of virus was detected, no infectious virus was detected in any of
the other fractions. The impact of fluctuations in virus concentration
was explored with Methods 2 and 3, where the mAb concentration was held
constant at the same level as used in Method 1. Virus stock was spiked
into a smaller feed pool and applied to the column as a pulse with a
significantly higher virus titer. For Method 2, a lower concentration of
virus was introduced mid-way through the column load, resulting in a
broader pulse of virus. Method 3 used a higher concentration of virus in
a smaller volume than used in Method 2 so that a high titer pulse of MMV
was introduced mid-way through the load. Virus was completely cleared in
all flow-through fractions and in the collected product pools. This is
discussed in more detail in Section 3.2. Fluctuations in both the mAb
and virus concentration were simulated in Method 4, which might be
representative of a connected process, as discussed in Section 3.3. In
this method, over 60\% of mAb was fed into the chromatography column
through a single mAb peak, which characterizes the case of elution from
an immediately upstream bind-and-elute chromatography unit. Virus was
only spiked into the small concentrated mAb pool and fed along with the
mAb peak as shown in Figure 1. Complete clearance was achieved in all
fractions, and the final LRV was similar to that obtained in the Method
1 batch approach. Large volume testing was used for the pooled
fractions, and because of the additional sensitivity afforded by
assaying additional volume of the pool, a small amount of virus was
detected. Virus can also be spiked and mixed in-line with the feed
stream instead of spiking directly into the mAb pool, and this was
evaluated in Method 5. In this method, a constant level of virus was fed
into the column from a separate MMV stock pool throughout the run. The
virus stock was diluted with process buffer and the flow rate of virus
stock was controlled with respect to the flow rate of the feed stream so
that the virus concentration in the mixed stream was comparable to the
virus concentration in the first method's spiked batch pool and without
causing significant dilution in the mAb feed. As with the other methods,
the total virus loaded onto the column was 8.0 log\textsubscript{10}
TCID\textsubscript{50}. The load peak of concentrated mAb, introduced in
Methods 4 and 5, simulates a load from the elution of a bind-and-elute
chromatography in a physically connected process. The viral clearance
performance of the AEX using in-line spiking method agreed well with
conventional batch spiking method. This is discussed in more detail in
Section 3.4. The impurity (HCP) removal using different methods was
summarized in Table 3. The total HCP in the load was calculated from the
sum of the mass of HCP in all feed pools. The total HCP in the flow
through pool was calculated based on the sum of the mass of HCP in all
pooled fractions. AEX demonstrates very robust performance for HCP
removal. Despite the variance in the feed and virus pattern loaded to
the column, over 95\% of HCP was removed from the feed; which was also
consistent with the AEX runs without any virus spiked in the load. AEX
(Eshmuno Q resin) can be run in flow through mode under protein load
density, pH and conductivity conditions where good separation of a
monoclonal antibody and virus is achieved. The data presented here also
demonstrate that when run under the same pH, conductivity and total
load, it is a robust unit operation despite significant differences in
mAb and virus loading patterns that might be observed in a continuous
process. The virus and impurity removal performance were very consistent
with different spiking and loading methods; at least 4.9
log\textsubscript{10} removal of MMV and a minimum of 95\% removal of
HCP were achieved for all methods. The study also suggests alternative
approaches to evaluate the viral clearance performance of a
chromatography unit in continuous processes. The conventional batch
validation method can be modified and applied without setting up the
physically connected system in a contract testing laboratory. Ionic
strength and pH peaks might also be created by splitting mAb feed in
pools comprised of different solution conditions, and spiking and
loading individual pools separately through gradient functions.

\subsection*{3.2 Effect of virus concentration on (AEX resin
capacity)}

{\label{470118}}

In order to determine the effect of variation in virus concentration on
virus removal by AEX, three sets of experiments (Methods 1-3) were
performed using the same total virus load but altering the virus
concentrations and in and the volume of the virus spike. The total virus
load for all three methods was maintained at 8.0 +- 0.2
log\textsubscript{10} TCID\textsubscript{50}. Virus was spiked into and
loaded from an 80 mL (20 CV), 8 mL (2 CV) or 1 mL feed pool
respectively. Although a similar total amount of MMV was spiked for
Methods 1-3, the virus concentrations for the three methods were
measured to be 6.3 +- 0.3, 6.9 +- 0.3 and 7.9 +- 0.3
log\textsubscript{10}TCID\textsubscript{50}/mL respectively. For all
chromatography runs using these load methods, the pH, conductivity,
protein load, flow rate and residence time were held constant at the
values that had been defined to achieve separation of this mAb and
virus. MMV was selected to demonstrate the feasibility of the study
because it typically represents a worst-case virus for viral clearance
(Gefroh et al., 2014). For conventional batch mode clearance studies,
virus is spiked into the mAb feed as a total virus load or based on a
volumetric proportion of the load (e.g., 1\% v/v). The amount of spiked
virus, especially when based on a volumetric proportion, cannot be too
high (typically less than 5\% v/v) or it may impact the physical and
chemical properties of the load material and consequently, the
performance of the AEX. Therefore, the total amount of virus that can be
added to the load material is limited by the virus titer of the stock.
By spiking the same amount of virus into a smaller load pool and loading
the entire virus pool at once, the impact of a bolus of high
concentration virus can be evaluated.

The viral clearance by AEX using these different ways of loading viruses
was consistent (LRV's of 5.5 log\textsubscript{10} or greater for all
runs). The binding capacity of Q resin was found to be significantly
higher than the level of virus loaded to the AEX step in a typical viral
clearance study. Strauss et al. (2009b) demonstrated that the binding
capacity for SV40 on Q Sepharose Fast Flow resin for a feedstock with a
conductivity of 7 mS/cm to be greater than 12.2
log\textsubscript{10}virus particles per mL of resin. Trilisky and
Lenhoff (2007) showed that the static binding capacity of Q Sepharose XL
resin for recombinant adenovirus type 5 (Ad5) reaches 12.9
log\textsubscript{10} particles/mL resin. Most virus particles bind to
the surface of the resins as the large size of virus restricts the
diffusion of virus particles into the small pores in the beads (Strauss
et al., 2009). The binding capacity of virus on AEX resin can be
affected by many factors including the size, surface chemistry,
isoelectric point (pI) of the virus particle, and the size and
composition of the resin backbone. Under the load conditions used in
this study, where the pH of the load was significantly higher than the
pI of MMV and the conductivity of the load was relatively low, it is
reasonable to assume that the binding capacity of MMV on the Eshmuno Q
resin is at least several orders of magnitude higher than the total
virus load (approximately 8.0 log\textsubscript{10} infectious
particles). Our study demonstrates that if the level of virus load is
significantly lower than the capacity limit of the Q resin, complete or
near complete removal of virus from the process stream is achieved,
regardless of the virus load concentration (up to 7.9 +- 0.1
log\textsubscript{10}/mL TCID\textsubscript{50}) or pattern of virus
loading.

The results also provide additional insight into the kinetics of virus
binding to the Q resin. The bed height and consequently, the linear flow
velocity were held constant when scaling down the manufacturing scale
column AEX step. The bed height and linear flow velocity were 300cm/hr
and 20cm respectively, which is typical for commercial manufacturing
processes for biologicals. Our results clearly suggest that over
99.999\% of virus can bind to Eshmuno Q resin with a 4 min average
residence time and at virus concentrations up to 7.9
log\textsubscript{10}/mL. The binding of virus to the resin is
determined by the diffusion of virus particles to the resin surface. The
time scale for diffusion can be estimated using Equation (1)

\(t_{D}\sim\frac{L^{2}}{D_{0}}\) (1)

where L is the diffusion distance and D\textsubscript{0} is the
diffusion coefficient (Bird et al., 2007). The diffusion coefficient for
MMV can be estimated as 1.6 x
10\textsuperscript{-7}cm\textsuperscript{2}/s, using the diffusion
coefficient found for human parvovirus B19 particles, due to similarity
in their sizes and shapes (Toivola et al., 2004). The extra-particle
flow channel is estimated to be about one third of the resin bead size
(Ingham \& Pop, 1998; Du Plessis \& Woudberg, 2008), which is close to
30\selectlanguage{greek}µ\selectlanguage{english}m for Eshmuno resins. The time scale for MMV particles to diffuse to
the resin surface is approximately 60 sec, which is much shorter than
the average residence time of virus particles. Therefore, under
appropriate conditions of pH and conductivity, viruses will have
sufficient time to bind to the resin surface, resulting in near complete
clearance.

\subsection*{3.3 Effect of fluctuations in feed on viral
clearance}

{\label{196212}}

AEX often follows a bind-and-elute cation exchange chromatography (CEX)
unit as a flow-through polishing step in continuous processes for
purification of monoclonal antibodies. The product is eluted from the
CEX column using a stepwise or gradient elution, and the mAb and most of
the impurities, including HCP and DNA, are contained in elution peaks
(Pabst et al., 2009; Stein and Kiesewetter, 2007; Yigzaw et al., 2009).
Depending on the load conditions, viruses either may not bind to the
negatively charged resin or are likely to be eluted as the salt
concentration or pH begin to increase. The virus elution profile may not
completely overlap with the mAb (Connell-Crowley et al., 2012; Miesegaes
et al., 2012). Nevertheless, co-elution of virus with mAb peaks
represents the worst-case scenario for both viral clearance and impurity
removal as all negatively charged species will compete for the
electrostatic binding sites when the mAb feed flows through the AEX
column.

To simulate an elution peak from the CEX unit, in Method 4, the same
amount of virus stock was spiked into a smaller mAb pool (2CV) as
described in Section 2.4. Although the total number of infectious viral
particles was the same for both Methods 1 and 4, the virus concentration
for Method 4 was measured to be 7.2 \selectlanguage{ngerman}± 0.3
log\textsubscript{10}TCID\textsubscript{50}/mL, which, due to the
smaller pool volume, was about tenfold higher than the virus
concentration of the batch load in Method 1. The load of spiked mAb pool
appeared as a sharp peak in the chromatogram with an average mAb
concentration of 56 g/L. The concentration of HCP also increased from
1039 ng/mL in the batch AEX pool to 8200 ng/mL in the concentrated mAb
pool, which was proportional to the increase in mAb concentration (data
not shown). The total HCP load for Method 4 was calculated as the sum of
HCP from the two mAb pools. The AEX step demonstrated robust performance
for both viral clearance and impurity removal, despite concentrated
pulses of impurities and virus. For Method 4, no infectious virus was
detected in any of the fractions (Figure 2), although when the fractions
were pooled and the sensitivity of the infectivity assay increased by
using large volume testing, a small amount of virus was detected.
Overall, 5.5 log\textsubscript{10} clearance was achieved (Table 2). The
LRV was very close to standard batch method, Method 1, also shown in
Table 2. The results also suggest that the batch viral clearance
experimental design can potentially be used to evaluate clearance for
the AEX flow through unit operating in a continuous process. Further
design of experiments (DOE) studies covering more extensive operational
parameters, including mAb loading, solution pH and conductivity, are
needed to support this conclusion.

\subsection*{3.4 Virus introduction by in-line
spiking}

{\label{962804}}

In-line virus spiking was initially developed to overcome challenges
associated with virus reduction filtration feed stocks that prove
difficult to filter. It allows the use of in-line prefiltration with
direct measurement of virus filter removal capabilities (Lutz et al.,
2011). This method can potentially be applied to more representative
scale down models of continuous manufacturing operations and can capture
the non-uniform conditions introduced by linked processes. In addition,
in-line spiking can be useful in situations in which protein forms
plugging foulants once virus is spiked into some mAb feeds. This
facilitates the evaluation of viral clearance for continuous processes,
since many of them might run for extended periods of time compared with
conventional batch processes.

A simple in-line spiking methodology was developed for chromatography
unit operation using the ÄKTA system. Virus stock was diluted with
process equilibrium buffer, pumped through a separate line and was mixed
with the mAb feed stream in-line using the chromatography system's mixer
before feeding to the AEX column. Fluctuation in the mAb concentration
was created as previously described. Before running the viral clearance
experiment, the in-line spiking method was first qualified. Two mAb feed
pools were prepared at low or high mAb concentration. The virus
concentration in the diluted virus pool was measured to be 7.2 ± 0.4
log\textsubscript{10} TCID\textsubscript{50}/mL. The predicted virus
concentration after in-line mixing was calculated from Equation (2)
based on steady-state mass balance:

\(C_{\text{sample}}=\frac{C_{v}\times Q_{v}}{C_{f}+Q_{v}}\) (2)

where C\textsubscript{sample} is the sample virus concentration at the
inlet of chromatography column, C\textsubscript{v} is the virus
concentration in the virus pool, Q\textsubscript{f} is the mAb feed flow
rate, and Q\textsubscript{v} is the diluted virus solution flow rate.
The flow rate of diluted virus stock was set to be 5\% of the total flow
rate so that it did not significantly impact the load properties, which
is confirmed by the chromatogram (data not shown). The results are
summarized in Table 4. The measured sample virus concentrations were
close to the predicted value for both concentrations of mAb feed, with
maximum errors less than 4\%. This demonstrates that the use of the ÄKTA
pumping and mixing system for the in-line spiking method delivers that
targeted concentration of virus.

Figure 2 and Table 2 demonstrate that the viral clearance achieved with
the in-line spiking method was very similar to the other methods
evaluated in this study. For this method and also Method 1, a small
amount of virus was detected in the first (Method 1) or first and second
(Method 5) fractions, while no virus was detected in the subsequent
fractions. These results are unexpected, because virus breakthrough
would only be likely once the column had become saturated and not in the
early fractions of the flow through. It is possible that virus was at
very low levels in all fractions so that virus may or may not have been
contained in each volume of sample that was assayed. It is possible that
by chance those samples assayed in those early fractions contained a
small amount of detectable virus, while the later fractions did not.

Since it may be hard to predict the pattern of virus levels that might
be expected in a connected process contaminated with virus, these data
support the use of a constant introduction of the spike using an in-line
method, at least for evaluation of flow-through chromatography steps.
These data also demonstrate that in-line spiking is a useful tool for
delivering a virus spike when mimicking the mAb feed loading patterns
that might be observed in a connected flow-through chromatography step.

\subsection*{4.~Conclusion}

{\label{386756}}

To ensure the viral safety of a continuous process, it is critical to
evaluate the viral reduction afforded by the manufacturing process.
Viral clearance studies require the use of representative small-scale
models (Johnson \& Roush, 2018), but models for continuous processes
typically require complex scaled down models, including multi-column
chromatography systems. The systems often vary from manufacturer to
manufacturer, making it difficult for the contract laboratories that
support viral clearance studies to provide this equipment. Data are
beginning to suggest that clearance studies, using standard batch models
of chromatography steps, may be predictive of the clearance that might
be achieved in a continuous process (Angelo, 2019). In this study, we
developed alternative spiking and loading methods to evaluate the viral
reduction of a flow-through AEX in the context of continuous processing
using a standard ÄKTA system. The studies were designed to mimic the
potential fluctuations in the feed material that might be introduced by
a connected, continuous process. Results suggest that AEX is robust in
viral and impurity removal despite loading high and fluctuating product
and virus concentrations. The study also demonstrates the design of a
batch clearance study representing a potential `worst-case scenario' in
continuous processing that can be readily adopted in a contract testing
lab. The advantage of this approach is that it can be used in evaluating
viral clearance by the AEX unit operating in a variety of different
continuous processes without setting up the complex physical system in
the testing lab each time. The methods developed in this study can be
easily extended to simulate other fluctuating conditions, such as pH,
buffer composition, salt concentration, multiple peaks of concentrated
mAb load or virus spike. The use of these load/spike methods for a
chromatography step that provides less robust clearance might also be
evaluated to determine whether an impact on clearance can be detected.
The methods can also be readily adopted in the evaluation of other
flow-through chromatography (such as membrane chromatography) that may
have fluctuations in feed introduced from a previous unit operation.

\textbf{Acknowledgements}

The authors thank Joe Orlando and Brandon Medeiros for generating
feedstocks, Elizabeth Goodrich, Akshat Gupta, Santosh Rahane for helping
prepare feedstocks, David Casaperalta, Dominic Smith, Alvaro Botero for
performing infectivity assays, Robert Kelly, Thomas Jefferies, David
Bohonak, Ushma Mehta, Herb Lutz and Tom Elich for valuable discussions.
We are grateful to Kate Smith for her careful review of the manuscript.

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\textbf{Figures}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure 1: Diagram of viral and mAb loading patterns of methods 1-5.
Total mAb loading was maintained at 175\selectlanguage{ngerman}±10g/L, and total virus loading
was maintained at approximately 8.0
log\textsubscript{10}TCID\textsubscript{50} for all runs. Solid lines
and dotted lines indicate mAb and virus concentration fed to the AEX
column, respectively.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2: Comparison of virus in flow through fractions collected from
an Eshmuno Q anion exchange chromatography column that had been loaded
and spiked using methods 1-5. The bars represent the total MMV remaining
in fractions 1-4 and the final buffer wash. Arrows indicate that no
detectable virus could be recovered. Although the virus spike was
delivered at different concentrations, approximately 8
log\textsubscript{10} TCID\textsubscript{50} MMV was loaded onto each
column. Each bar represents the mean of two runs.

\textbf{Tables}

Table 1: The different loading and spiking conditions used in methods
1-5. Experiments were performed in product flow-through mode in Tris-HCl
buffer (pH 8.5 and conductivity 6 mS/cm), residence time were maintained
at 4 min for all runs. Although the virus may have been more or less
concentrated by the different loading methods, each run was spiked with
a total of approximately 8 log\textsubscript{10}
TCID\textsubscript{50}MMV. The data represent the mean of two runs.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\emph{*Virus was not spiked in mAb pools for Method 5, but continuously
introduced during the entire load. The virus concentration was estimated
to be 6.1 log\textsubscript{10}/mL TCID\textsubscript{50}}

\emph{NA -- not applicable}

Table 2: Viral clearance by Eshmuno Q anion exchange chromatography
using different loading and spiking methods. Experiments were performed
in product flow-through mode in Tris-HCl buffer (pH 8.5 and conductivity
6 mS/cm); residence time was maintained at 4 min for all runs. Each run
was spiked with a total of approximately 8
log\textsubscript{10}TCID\textsubscript{50} MMV. The flow through pool
represents the virus present in the pooled four flow through fractions
and wash fraction. The data represent the mean of two runs.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Table 3: Impurity removal by Eshmuno Q anion exchange chromatography
using different loading and spiking methods. Experiments were performed
in product flow-through mode in Tris-HCl buffer (pH 8.5 and conductivity
6 mS/cm), residence time were maintained at 4 min for all runs. MMV was
used for spiking.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Table 4: In-line qualification with \selectlanguage{ngerman}ÄKTA pumping and mixing system. Feed
pools were prepared with same mab stock in Tris-HCl buffer (pH 8.5 and
conductivity 6 mS/cm).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\selectlanguage{english}
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