1. Introduction
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.
2. Materials and Methods
2.1 Feed stocks
Feed material was a monoclonal antibody (IgG1) generated internally
by MilliporeSigma (Bedford, MA). The harvested cell culture was
purified by Clarisolve® Pod (MilliporeSigma/Merck
KGaA, Darmstadt, Germany) clarification, Eshmuno®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 µm
filter prior to use.
2.2 Virus Stocks and Assays
Mouse minute virus (MMV) stocks were provided by MilliporeSigma
(Rockville, MD). A single lot of MMV with a certified titer of 8.6 ± 0.3
log10 TCID50/mL was used for all runs.
The MMV titer for each test sample was quantified by 50% tissue culture
infectious dose (TCID50) 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. Log10 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 A280absorbance, using an extinction coefficient of 1.47. Residual CHO host
cell proteins (CHO-HCP) were measured by CHO ELISA kits (Cygnus
Technologies, Southport, NC).
2.3 Anion exchange chromatography
Chromatography columns (0.5cm diameter, 20cm bed height, 4mL column
volume, OPUS® ValiChrom™) prepacked with
Eshmuno® 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 ÄKTA
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
OD280 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.
2.4 Viral spiking and loading methods
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 log10TCID50.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 log10TCID50/mL (a total of 8.2 log10TCID50 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.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 log10TCID50. 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 log10TCID50/mL, mid-way through the loading of the mAb.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 log10 TCID50; the
concentration was 7.9 ± 0.3 log10TCID50/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.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 log10TCID50; the concentration of virus was 7.2 ± 0.3
log10 TCID50/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.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 log10 total
TCID50) 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 ÄKTA 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.
3. Results and discussion
3.1 Viral and HCP clearance by different load and spike methods
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 log10 TCID50 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 log10 TCID50. 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 log10 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.
3.2 Effect of virus concentration on (AEX resin capacity)
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
log10 TCID50. 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 log10TCID50/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 log10 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 log10virus 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 log10 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 log10 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
log10/mL TCID50) 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
log10/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 D0 is the
diffusion coefficient (Bird et al., 2007). The diffusion coefficient for
MMV can be estimated as 1.6 x 10-7cm2/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 30µm 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.
3.3 Effect of fluctuations in feed on viral clearance
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 ± 0.3
log10TCID50/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 log10 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.
3.4 Virus introduction by in-line spiking
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
log10 TCID50/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 Csample is the sample virus concentration at the
inlet of chromatography column, Cv is the virus
concentration in the virus pool, Qf is the mAb feed flow
rate, and Qv 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.
4. Conclusion
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.