Strategies for Developing Design Spaces for Viral Clearance by Anion Exchange

Chromatography During Monoclonal Antibody Production

Daniel M. Strauss, Tony Cano, Nick Cai, Heather Delucchi, and Magdalena Plancarte
Process Research and Development, Genentech, Inc., One DNA Way, South San Francisco, CA 94080

Daniel Coleman
Non-Clinical Biostatistics, Genentech, Inc., One DNA Way, South San Francisco, CA 94080
Gregory S. Blank, Qi Chen, and Bin Yang
Process Research and Development, Genentech, Inc., One DNA Way, South San Francisco, CA 94080

DOI 10.1002/btpr.385
Published online January 29, 2010 in Wiley InterScience (www.interscience.wiley.com).

The quality-by-design (QbD) regulatory initiative promotes the development of process

design spaces describing the multidimensional effects and interactions of process varia-
bles on critical quality attributes of therapeutic products. However, because of the com-
plex nature of production processes, strategies must be devised to provide for design
space development with reasonable allocation of resources while maintaining highly de-
pendable results. Here, we discuss strategies for the determination of design spaces for
viral clearance by anion exchange chromatography (AEX) during purification of monoclo-
nal antibodies. We developed a risk assessment for AEX using a formalized method and
applying previous knowledge of the effects of certain variables and the mechanism of
action for virus removal by this process. We then use design-of-experiments (DOE) con-
cepts to perform a highly fractionated factorial experiment and show that varying many
process parameters simultaneously over wide ranges does not affect the ability of the
AEX process to remove endogenous retrovirus-like particles from CHO-cell derived feed-
stocks. Finally, we performed a full factorial design and observed that a high degree of
viral clearance was obtained for three different model viruses when the most significant
process parameters were varied over ranges relevant to typical manufacturing processes.
These experiments indicate the robust nature of viral clearance by the AEX process as
well as the design space where removal of viral impurities and contaminants can be
assured. In addition, the concepts and methodology presented here provides a general
approach for the development of design spaces to assure that quality of biotherapeutic
products is maintained. V C 2010 American Institute of Chemical Engineers Biotechnol.

Prog., 26: 750–755, 2010

Keywords: quality-by-design (QbD), design space, viral clearance, anion exchange
chromatography (AEX), design of experiments (DOE)

Introduction cess knowledge required for approval of a QbD filing

Because biotherapeutic products are complex and difficult
to fully characterize, the process by which they are created
is an important factor in defining the final drug product. As
such, the characterization and validation of the processes are
critical parts of ensuring that products are consistently safe
and effective for patients. Quality-by-design (QbD) is a regu-
latory initiative that encourages a thorough understanding of
the process variables, which affect the quality of the product
and how those parameters interact with one another.1 Once
approved, the QbD approach may then allow for more regu-
latory flexibility in manufacturing process changes. The pro-
Current address of Daniel M. Strauss: Bioproduct Research and
Development, Eli Lilly and Company, Indianapolis, IN 64221
Correspondence concerning this article should be addressed to
B. Yang at yang.bin@gene.com.
involves the formation of a design space, which is defined as
‘‘the multidimensional combination and interaction of input
variables and process parameters that have been demon-
strated to provide assurance of quality.’’1 As long as changes
are made within the design space, the change can be dealt
with by internal quality controls rather than requiring regula-
tory approval. A general guidance for the development of
design spaces has been provided,1 but few examples of their
development in biotechnology settings have been
described.2–4 Additional case studies of design space devel-
opment will provide the industry with the concepts needed
to build systematic methods and take full advantage of the
QbD regulatory initiative.
The first step in the QbD process involves determining
critical quality attributes (CQAs), which are those attributes
of a product that characterize its quality, safety, and efficacy.
Although many products may have distinct CQAs, viral

750 C 2010 American Institute of Chemical Engineers

V
Biotechnol. Prog., 2010, Vol. 26, No. 3 751

Table 1. LRV Values Obtained from Small-Scale Virus Reduction Experiments

mAb1-a* mAb1-b mAb2-a mAb2-b mAb3-a mAb3-b mAb4 mAb5 mAb6 mAb7
XMuLV 3.7†,‡ 6.0 3.5‡ 5.1 5.8 5.4 6.2 5.5 5.7 5.7
SV40 5.8 4.8 5.1 4.1 4.7 4.0 5.7 4.6 4.3 4.1
MMV 5.5 5.3 5.0 5.4 5.0 5.7 5.3 4.7 5.5 5.2
Product mAb8 mAb9 mAb10 mAb11 mAb12 mAb13 mAb14
XMuLV 5.7 5.4 5.9 5.4 6.1 5.3 5.9
* Letters represent multiple processes developed at different scales for the same mAb product.
†
‘‘’’ symbols indicate that no virus was detected in the product pool.
‡
These values were obtained using infectivity assays, while all others were obtained using QPCR assays.

safety should be considered for all products, which are pro- Table 2. RVLP Design Space Parameters
duced in mammalian cell culture. These processes are known Parameters Low Center High
to produce endogenous retroviruses or retrovirus-like par-
Feedstock conductivity (mS/cm) 5.0 9.5 14.0
ticles (RVLPs),5–7 and they have the potential to be infected
Load density (mg/mL resin) 50 N/A* 200
by adventitious viruses during mAb production.8 Any viral Feedstock pH 7.0 7.8 8.5
impurities that are or could be present in the product pools Equil. buffer conductivity (mS/cm) 5.0 9.5 14.0
must be removed or inactivated by the product purification Feedstock impurity level No Varied HCCF
process.9–11 One unit operation that plays an important role spike spike
Buffer pH 7.0 7.8 8.5
in providing acceptable levels of the viral clearance CQA is Temperature ( C) 15 23 30
the anion exchange chromatography (AEX) step. AEX per- Flow rate (cm/h) 50 150 250
formed in product flow-through mode has been shown to be Equil. buffer tris-concentration (mM) 15 25 35
capable of providing a high level of removal of both nonen- Equilibration phase duration (CV) 3 5 7
veloped and enveloped viruses, including the endogenous * Both load density values were obtained for all runs.
RVLPs that are produced by CHO-cells.12–14 This process
has also been shown to be very robust. It has been tested (SevenMulti, Mettler Toledo, Columbus, OH), and either
over wide ranges of parameters and shown to consistently WFI or sodium acetate was added to achieve the desired
provide log reduction values greater than 4.15,16 In addition, conductivity. Selected loads were also spiked to 0.1% V/V
our group has validated this process for many different mAb with harvested cell culture fluid (HCCF) to introduce
products and processes (Table 1). In any case, no specific increased levels of host cell impurities. The feedstock was
product effect has been observed, and despite many differen- then filtered through a 0.45 lm PVDF filter (Millipore, Bill-
ces between those processes, the step has consistently pro- erica, MA), and the protein concentration was determined by
vided excellent LRV values. The robustness observed for optical density at 280 nm (OD280).
viral clearance by AEX makes it an excellent candidate for
A small-scale chromatography column (0.66-cm diameter,
the application of QbD concepts.
Bio-Chem Valve/OmniFit, Boonton, NJ) was packed with
In this work, we discuss strategies for developing design naive QSFF resin to 20-cm bed height. Chromatography runs
spaces for viral clearance and present examples of how we were performed with varying parameters as described in
have applied these concepts to AEX processes. We first de- Table 2. Column equilibration was first performed using tris-
velop a formalized risk assessment to cover multiple AEX acetate equilibration buffers with varied compositions and
processes by using previous knowledge of the effects of pa- for varied durations. The feedstock pool was then loaded
rameters on viral clearance as well as the mechanism of onto the column, and collection of the flow-through fraction
action of the process. We then present experimental meth- was started when the OD280 reached 0.1 unit above baseline.
ods, which utilize that risk assessment as well as DOE tech- Protein was loaded to a total of 200 g mAb/mL resin, chang-
niques, to develop an understanding of process variability on ing the collection vessel after the initial 50 g mAb/mL resin
viral clearance. Using a highly fractionated experimental was loaded and allowing for analysis of both load densities
design, we show that the AEX can provide complete re- from each chromatography run. The column was then
moval of the endogenous RVLPs present in CHO-cell washed with three column volumes (CV) of equilibration
derived feedstocks over wide ranges of many process param- buffer, followed by regeneration with four CV of 0.5 N
eters. Finally, we present a full factorial design space in NaOH and three CV of 0.1 N NaOH. Aliquots of the product
which very high LRV values are obtained for three different pools and remaining feedstock pools were collected and
model viruses for all combinations of parameters within stored at 80 C. Extraction of viral genomic material was
ranges typical of AEX manufacturing processes. performed either using the Virus Mini Kit v2.0 on a BioRo-
bot EZ1 (Qiagen, Valencia, CA) or the MagAttract Virus
Materials and Methods Mini Kit v1.3 on a BioRobot M48 (Qiagen, Valencia, CA).
In both cases, extractions were followed by treatment with
RVLP design space RNase-free DNaseI (Strategene, Santa Clara, CA) to remove
Stock monoclonal antibody intermediate protein A pools any contaminating CHO-cell genomic DNA, and QPCR
were obtained from the mAb15 commercial-scale manufac- assays were performed as described previously.17
turing operations. For each chromatography run, pool condi-
tions were adjusted to varying levels as indicated in Table 2.
The pool was first adjusted to the appropriate pH using tris- Model virus design space
base or HEPES acid. The conductivity of each pool was then Stock monoclonal antibody intermediate cation chroma-
measured using a temperature-adjusted conductivity meter tography pools were obtained from mAb1 commercial-scale
752
manufacturing operations. For each chromatography run,
load pools were conditioned to varying levels as indicated in
Table 4. The pool was first adjusted to the appropriate pH
using 1.5 M tris-base. The conductivity of each pool was
then measured using a temperature-compensated conductivity
meter (SevenMulti, Mettler Toledo, Columbus, OH), and ei-
ther WFI or 5 M NaCl was added to adjust the conductivity
to the desired level. The feedstock was then filtered through
a 0.22 lm PES filter, and the protein concentration was
determined by optical density at 280 nm (OD280).
For each run, a small-scale chromatography column (0.66-
cm diameter, Bio-Chem Valve/OmniFit) was packed with
naive QSFF resin to 20 cm bed height and equilibrated with
eight CV equilibration buffer (25 mM tris, NaCl, pH and
conductivity adjusted to match load pool). The feedstock
pool was then loaded onto the column, and collection of the
flow-through fraction was started when the OD280 reached
0.1 unit above baseline. The feedstocks were loaded at vary-
ing flow rates and to varying load densities as indicated in
Table 4, and the columns were then washed with three CV
equilibration buffer. On completion of the chromatography
run, the protein concentration of the product pool was deter-
mined by OD280, and aliquots of this pool as well as the
remaining feedstock pool were collected and stored at
80 C. The samples were subject to treatment with DNaseI
(Applied Biosystems, Foster City, CA), followed by extrac-
tion of viral genomic material using the EZ1 Virus Mini Kit
v2.0 on a BioRobot EZ1 (Qiagen, Valencia, CA). QPCR
assays used to quantify X-MuLV, SV40, and MMV viral
particles were performed as previously described.13,18,19
Results and Discussion
Risk assessment
As a multidimensional study of input variables, design
space experiments have the potential to be quite extensive.
When performed in product flow-through mode during mAb
production, AEX is relatively straightforward compared with
other bind-and-elute chromatography steps.20 However, we
have identified 18 sources of variability for this unit opera-
tion. Using two-level screening designs to test variables at
the high and low ends of a range, full factorial experiments
to investigate all combinations require at least 2n runs, where
n is the number of parameters. For AEX, testing all combi-
nations of the 18 variables identified would require over
260,000 runs, an unfeasible number, especially for viral
clearance experiments, which, due to the high costs of virus
stocks, are generally expensive to perform. Therefore, we
utilized two tools to reduce the number of runs required to
perform design space studies: development of a formalized
risk assessment and utilization of design-of-experiments
(DOE) techniques.
The goal of a risk assessment is to provide an unbiased
ranking of variables so that experiments can be devised that
focus on the factors most likely to have an impact on pro-
cess performance or have significant interactions with other
factors. Because of the robust nature of viral clearance by
AEX with regard to both products and process parameters,
we focused on constructing a risk assessment based on the
ranges of variables from the AEX processes for many differ-
ent mAb products. These processes are often optimized on a
product-specific basis for yield and impurity removal, and
many parameters can vary significantly between products.
Biotechnol. Prog., 2010, Vol. 26, No. 3
This strategy led to increased parameter ranges and consider-
ation of many categorical parameters, such as buffer and salt
compositions, which are generally locked for a specific pro-
cess but are varied between different processes. We used a
variation of an FMEA risk assessment tool, taking into
account the severity that variability in a parameter may have
on viral clearance and the probability that the parameter
would be variable. The effects of each factor were first ana-
lyzed using previous data, which indicated their effects on
viral clearance. For instance, our laboratory has previously
determined that feedstock conductivity, pH, and salt compo-
sition can have effects on LRV values, and they interact
with one another,15,16 whereas variables such as the buffer
compositions and pooling criteria were not observed to
impact viral clearance. In addition, while considering the
effects of certain parameters, we also took into account the
mechanism of viral clearance by this process. The binding
interactions that allow removal of viruses from mAb prod-
ucts are primarily due to electrostatic forces.21 This knowl-
edge emphasizes the impact that variability in factors, which
are related to electrostatic forces, such as conductivity and
pH, may have on viral clearance. In addition, the mechanism
suggests that impurities that compete for the electrostatic
binding sites, such as anion host cell proteins, may also
impact viral clearance. This observation increases the impact
of parameters such as impurity level, as well as load density
since it relates directly to total impurity load. For each pa-
rameter, we also gauged the probability that it would vary
between different processes by comparing the many AEX
processes previously developed in-house. The results of our
risk assessment indicate that feedstock conductivity and load
density are the parameters, which are most likely to impact
viral clearance by AEX, while feedstock pH, impurity levels,
and buffer conductivity also may have some impact (Figure
1). This risk assessment provides a useful tool for comparing
the viral clearance ability of different processes or predicting
the effects of process changes. Furthermore, the strategy pre-
sented for the formation of a risk assessment provides the
opportunity to apply previous knowledge and experience
with a process to guide the development of design space
experiments.
Fractionated design space for RVLP clearance
Another way to reduce the overall number of runs neces-
sary to explore a design space with many parameters is
through the use of DOE experiments. DOE techniques
ensure that the efficiency of an experiment matches the
desired outcome, maximizing reliable information gained
while minimizing redundancy between runs. For initial
design space experiments, two-level screening designs are
well-suited to give information on which parameters have
main effects on the desired outcome, and often give informa-
tion as to the interactions of those variables. For a given
number of runs, DOE approaches range from highly fractio-
nated designs to nonfractionated full factorial designs.
Highly fractionated designs can test many different parame-
ters, providing information on main effects of most parame-
ters but only providing interactions between a few. Because
many parameters can be tested with these designs, they rely
less on the risk assessment to choose which parameters
should be tested. However, because many parameters are
adjusted for each run, the experiments can be logistically dif-
ficult, and since the results contain more information than
Biotechnol. Prog., 2010, Vol. 26, No. 3 753

Table 3. LRV Values from Fractional Factorial Design Space of

RVLP Removal
Load Density (g/L resin)
Run 50 200
1.* 3.7 3.7
2.* 3.5 3.5
3 3.3 3.3
4 3.3 3.3
5 3.4 3.4
6 3.4 3.4
7 3.3 3.3
8 2.9 2.9
9 3.2 3.2
10 3.2 3.2
11 3.2 3.2
12 3.2 3.2
13 3.2 3.2
14 3.2 3.2
15 3.0 3.0
16 3.0 3.0
17 3.0 3.0
18 3.0 3.0
19.* 3.4 3.4
20.* 3.5 3.5
* Center point runs.

Table 4. Model Virus Design Space Parameters

Figure 1. Risk assessment for viral clearance by AEX per-
formed in product flow-through mode.
Process variables for a QSFF step performed in mAb flow-
through mode are listed in the order of highest risk priority to
lowest risk priority with bars indicating the magnitude of the
risk priority numbers associated with each factor.
the familiar one-factor-at-a-time experiments, some research-
ers and regulatory personal may require the assistance of a
statistician to help in analysis and interpretation of the data,
particularly when using designs with complex aliasing struc-
tures. Full factorial designs, on the other hand, provide both
main effects and high-level interactions for all parameters.
For a given number of runs, fewer parameters can be tested
with these designs, so although they rely heavily on the risk
assessment in choosing the most important factors, they are
logistically easier to carry out. In addition, since all combi-
nations of all the included parameters are tested, it is easy to
grasp the full meaning of the resulting data. Our experimen-
tal strategy included an iterative approach, including a highly
fractionated design to test many of the AEX parameters, re-
evaluation of the risk assessment based on the outcome of
that data, and then a full factorial design to test the most sig-
nificant parameters.
We first investigated the effects of many AEX variables
using a highly fractionated two-level screening design. To
minimize the cost associated with this preliminary experi-
ment, we chose to test removal of in-process RVLPs using a
protein A pool as a feedstock. This methodology allows for
the determination of LRV values by the AEX process with-
out the need for expensive virus spiking, although the maxi-
mum LRV values which can be obtained are limited by the
starting RVLP titers. We used a fractional factorial design to
test nine different parameters over large ranges. The design
consisted of 16 runs plus four center point runs, and the runs
were grouped to form a split plot design with similar feed-
Parameters Low Center High
Conductivity* (mS/cm) 5.5 7.3 9.0
Load density (mg/mL resin) 10 80 150
pH* 7.5 8.0 8.5
Flow rate† (cm/h) 43 115 186
* Conductivity and pH of feedstock and buffer were matched.
†
Accounts for contact time variation from production variations in
both flow rate and bed height.
stocks and column temperature runs performed in pairs,
allowing two runs per day. In addition, we collected multiple
flow-through pools for each run allowing for LRV values to
be calculated for different load densities. This design strat-
egy allowed for 10 of the 18 AEX variables to be varied
(Table 2), while two others, feedstock and buffer salt compo-
sitions, were held at worse case conditions for viral clear-
ance.16 We observed that virus titers in all of the product
pools were below the limit of quantification (LOQ) for the
QPCR assay, indicating that all 20 runs achieved complete
or near-complete removal of RVLPs for all load densities
tested. LRV values for the runs, which were calculated from
the input titers and QPCR LOQ values (Table 3), varied
from 2.9 to 3.7, with variations primarily stemming from
the QPCR assays of the feedstocks. Since complete removal
was observed for all of the runs, the data cannot be used to
determine the effects of the varied chromatography parame-
ters. Re-evaluation of the risk assessment based on these
data did not result in changes to the previous order of
parameters. However, the data does indicate that variation of
even the most important AEX variables does not have a
measurable impact on its ability to remove RVLPs. Further-
more, the robust removal of RVLPs over these broad ranges
of AEX parameters establishes a minimal design space
where consistent RVLP removal can be achieved by this
process.
Full factorial design space for model virus clearance
The design space from the first experiment provides strong
evidence that viruses can be removed over wide ranges of
754 Biotechnol. Prog., 2010, Vol. 26, No. 3

Table 5. LRV Values from Full Factorial Design Space of Model mammalian-cell derived mAb products. Through the system-
Virus Removal atic development of a risk assessment and two carefully
Model Virus designed experiments, we have shown that the AEX process
Run X-MuLV SV40 MMV can remove the endogenous RVLP impurities over ranges of
1.* 5.9 5.4 5.7
parameters much wider than those typically found in produc-
2 5.7 4.9 5.4 tion processes, and we have provided ranges of process vari-
3 5.8 5.4 5.5 ables where AEX can also be assured of providing complete
4 6.1 5.5 5.7 or near-complete removal of potential adventitious virus
5 5.7 5.5 5.7 contaminants.
6 5.8 5.1 5.5
7 5.7 5.3 5.5
8 6.0 5.6 5.8
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