RESEARCH ARTICLE – Pharmaceutical Biotechnology

Application of a Quality by Design Approach to the Cell Culture

Process of Monoclonal Antibody Production, Resulting in the
Establishment of a Design Space
HIROAKI NAGASHIMA, AKIKO WATARI, YASUHARU SHINODA, HIROSHI OKAMOTO, SHINYA TAKUMA

API Process Development Department, Chugai Pharmaceutical Company, Ltd., Tokyo, Japan

Received 11 August 2013; revised 9 September 2013; accepted 13 September 2013

Published online 3 October 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23744

ABSTRACT: This case study describes the application of Quality by Design elements to the process of culturing Chinese hamster ovary
cells in the production of a monoclonal antibody. All steps in the cell culture process and all process parameters in each step were
identified by using a cause-and-effect diagram. Prospective risk assessment using failure mode and effects analysis identified the following
four potential critical process parameters in the production culture step: initial viable cell density, culture duration, pH, and temperature.
These parameters and lot-to-lot variability in raw material were then evaluated by process characterization utilizing a design of experiments
approach consisting of a face-centered central composite design integrated with a full factorial design. Process characterization was
conducted using a scaled down model that had been qualified by comparison with large-scale production data. Multivariate regression
analysis was used to establish statistical prediction models for performance indicators and quality attributes; with these, we constructed
contour plots and conducted Monte Carlo simulation to clarify the design space. The statistical analyses, especially for raw materials,
identified set point values, which were most robust with respect to the lot-to-lot variability of raw materials while keeping the product
quality within the acceptance criteria. C 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:4274–

4283, 2013
Keywords: antibody; biotechnology; cell culture; design of experiments; design space; failure mode and effects analysis; mathematical
models; multivariate analysis; proteins; quality by design

INTRODUCTION efficacy and safety is necessary to achieve such pharmaceutical

development.
Quality by Design (QbD) is a science- and risk-based approach
The QbD approach is a more scientific, systematic, and com-
to pharmaceutical development and manufacture applicable to
prehensive approach that builds quality into the process in-
all stages of a product’s lifecycle.1,2 The QbD approach for bio-
stead of the traditional process of testing the quality of the
pharmaceutical manufacturing processes had its beginnings in
product throughout the biopharmaceutical’s lifecycle (e.g., de-
2002 with the Current Good Manufacturing Practices for the
velopment, manufacture). It is said that pharmaceutical com-
21st Century initiative launched by the United States Food
panies can understand their products more deeply and proceed
and Drug Administration,3 and the QbD concepts introduced
with development more flexibly and robustly through a QbD
there were subsequently followed by the International Con-
approach, and if the development and manufacture of biophar-
ference on Harmonisation of Technical Requirements for Reg-
maceuticals are carried out according to QbD elements using
istration of Pharmaceuticals for Human Use (ICH) guidance
appropriate tools, both pharmaceutical companies and regula-
documents.4–7 The traditional approach to pharmaceutical de-
tory agencies will be able to reap the resulting benefits.8 For
velopment has been a rigid manufacturing process producing
example, the design space for a manufacturing process is one of
a product of variable quality that can be controlled by speci-
the most important components in the QbD approach. The ICH
fications; that approach is being replaced by an enhanced ap-
guidance documents define design space and working within
proach based on QbD elements. Biopharmaceutical manufac-
the design space is not considered as a change. Some of the
turing processes must be developed to produce a product that
expected benefits of QbD for the biopharmaceutical industry
above all meets the needs of the patients while maintaining
thus include a reduction in the number of postapproval pro-
consistent efficacy and safety. Deep understanding of the re-
cess changes and an increase in flexibility for pharmaceutical
lationships among process parameters, quality attributes, and
companies.9,10
Interpretations of the application of QbD described in the
Abbreviations used: CE-SDS, capillary electrophoresis-sodium dodecyl sulfate;
CEX, cation exchange chromatography; CHO, Chinese hamster ovary; CPP, guidelines differ within the industry, and concrete case studies
critical process parameter; CQA, critical quality attribute; DoE, design of ex- of QbD as applied to biopharmaceutical drug substances are
periments; FMEA, failure mode and effects analysis; HPLC, high-performance
liquid chromatography; IVCD, initial viable cell density; mAb, monoclonal anti-
still limited. Some reports show original QbD approaches that
body; QbD, quality by design; RPN, risk priority number; TOST, two one-sided focus on risk assessment and process characterization using de-
t-test; VCD, viable cell density. sign of experiments (DoE) for the processes of cell culture11–20
Correspondence to: Hiroaki Nagashima (Telephone: +81-3-3968-6115;
Fax: +81-3-3968-6259; E-mail: nagashimahra@chugai-pharm.co.jp) and purification,13,15,21,22 and the CMC Biotech Working Group
Journal of Pharmaceutical Sciences, Vol. 102, 4274–4283 (2013) published a mock case study covering all of the QbD elements

C 2013 Wiley Periodicals, Inc. and the American Pharmacists Association for an antibody drug substance and drug product.23 Interpre-

4274 Nagashima et al., JOURNAL OF PHARMACEUTICAL SCIENCES 102:4274–4283, 2013


tation of these initiatives by industry has been an ongoing
process, with some notable industry and regulatory collab-
orations for biopharmaceuticals, and each pharmaceutical
company should take on the challenge of this approach and
find its own way.24,25
This study shows our QbD approach as applied to the process
of culturing Chinese hamster ovary (CHO) cells in the produc-
tion of a monoclonal antibody (mAb). The aim of this QbD case
study is to understand the cell culture process more deeply
and to increase the sophistication of the QbD approach itself.
The QbD approach has four important elements—identification
of critical process parameters (CPPs), identification of critical
quality attributes (CQAs), understanding the relationships be-
tween CPPs and CQAs, and establishment of an appropriate
control strategy. In this case study, we focused on quality risk
management using failure mode and effects analysis (FMEA),
and we conducted a multivariate study using DoE to establish
a design space.
Risk assessment was composed of risk identification, risk
analysis, and risk evaluation; it was carried out for the cell
culture process by using FMEA on the basis of established
scientific knowledge and our experimental knowledge. FMEA
is a common and useful risk analysis tool to find process pa-
rameters that require further process characterization.12,13,16
Process characterization experiments are typically conducted
using DoE to evaluate not only the main effects of parameters
but also their interactions.11–20 The potential CPPs that were
identified by this risk assessment of the production culture step
in the cell culture process were experimentally evaluated on a
scaled down model by a multivariate study using DoE. A design
space was established on the basis of those results and checked
by Monte Carlo simulation. Because lot-to-lot variability of raw
materials is said to contribute greatly to product quality, safety,
and process performance,26–28 our multivariate study included
an evaluation of material variability by finding the interac-
tions between material lot and process parameters. The scaled
down model used for process characterization was confirmed
to be appropriate by demonstrating that its performance was
comparable to that of a 2500 L bioreactor.
Through this QbD case study, we were able to understand
the cell culture process over a range of operation wider than
that traditionally used. In this article, we present the QbD
approach we have developed for the cell culture process of a
mAb currently under clinical development.
MATERIALS AND METHODS
Cell Culture
A CHO cell line expressing a glycosylated mAb was used in
this case study. Cells were grown in suspension using a serum-
free cell culture medium. Cells were subcultured using spinner
flasks (Bellco Glass, Vineland, New Jersey) in a CO2 incubator
(Thermo Scientific, Waltham, Massachusetts). Production cul-
ture was performed in fed-batch mode with continuous feeding.
We used 1 L bioreactors (ABLE, Tokyo, Japan) as the scaled
down model for process characterization, and a 2500 L biore-
actor (Hitachi, Tokyo, Japan) for the qualification of the scaled
down model. Cell culture fluids were centrifuged and filtered to
remove cells for analysis, and antibodies were purified from the
fluids with a POROS A20 protein A column (Life Technologies,
Carlsbad, California).
DOI 10.1002/jps.23744
RESEARCH ARTICLE – Pharmaceutical Biotechnology 4275
Analytical Methods
Viable cell density (VCD) and viability were measured by using
a Cedex automated cell counter and analyzer (F. Hoffmann-La
Roche, Basel, Switzerland). Offline pH was measured by us-
ing a GASTAT blood gas analyzer (Techno Medica, Kanagawa,
Japan). Antibody titer was measured by using protein A affinity
high-performance liquid chromatography (HPLC). Charge vari-
ants were measured by using cation-exchange chromatography
(CEX) HPLC. N-linked glycosylation (e.g., high-mannose) was
measured by hydrophilic interaction chromatography HPLC
after cutting with N-Glycosidase F (F. Hoffmann-La Roche) and
labeling with 2-aminobenzamide (Sigma–Aldrich, St. Louis,
Missouri). Whole antibodies (H2 L2 ) were measured by capillary
electrophoresis-sodium dodecyl sulfate (CE-SDS) using the PA
800 plus Pharmaceutical Analysis System (Beckman Coulter,
Brea, California).
Risk Assessment and Process Characterization
A cause-and-effect diagram was used for risk identification, and
all process steps and process parameters in the cell culture pro-
cess were defined. The method applied to the risk analysis and
risk evaluation of process parameters in the cell culture process
was based on FMEA. In this case study, two study designs gen-
erated by DoE were integrated and used for the process char-
acterization. The parameter IVCD, pH, and temperature were
evaluated by face-centered central composite design, and cul-
ture duration and material lot were evaluated by full factorial
design. The process characterization study was performed with
a total of 30 conditions. For the purposes of this case study, titer
was chosen as a representative key performance indicator and
the main peak in CEX, H2 L2 in CE-SDS, and high-mannose
were chosen as representative CQAs with which to analyze
the impact of process parameters statistically. Multivariate re-
gression analysis was performed for each performance indi-
cator and quality attribute to establish mathematical models.
Main effects and interaction effects of continuous factors on
responses were considered statistically significant when the F
ratio—which means the F value of the factor compared with
the F value of error—was more than 2.0. If a model was judged
as rational from statistical and scientific views, contour plots
were generated and superimposed to define the design space.
Monte Carlo simulation was performed to discover the distri-
bution of model outputs as a function of random variation in
the parameters and model noise. To evaluate the differences
arising from lot-to-lot variability in raw material, the main ef-
fect of material lot and the interaction effects between material
lot and the other parameters were also incorporated into the
prediction models.
Qualification of Scaled Down Model
Culture performance indicators and quality attributes of the
scaled down model were compared with those of the large-scale
production bioreactor to demonstrate the appropriateness of
the scaled down model. We compared data from 7 runs of the
scaled down model bioreactor, which ran during process devel-
opment as a control with data from 6 runs of the 2500 L biore-
actor used for the production of the clinical drug substance. The
cells used for small-scale culture were derived from the same
working cell bank as used for large-scale production. Five cell
tubes were used for the small-scale runs and two cell tubes were
used for the large-scale runs. Performance indicators, such as
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4276 RESEARCH ARTICLE – Pharmaceutical Biotechnology

Figure 1. A part of the cause-and-effect diagram identifying the process steps and associated process parameters in the cell culture process.
All identified process parameters were evaluated for further risk assessment. CQA: critical quality attribute, KPI: key performance indicator.

cell growth and titer, and quality attributes, such as CEX vari-
ants and whole antibody, were compared between the scales.
Comparability between the scaled down model and the large-
scale bioreactor was judged by using the mean ± 3SD of each.
RESULTS AND DISCUSSION
Risk Assessment for Process Parameters
Quality risk assessment comprises risk identification, risk
analysis, and risk evaluation. A cause-and-effect diagram—also
called a fishbone diagram or Ishikawa diagram—is commonly
used in many fields for risk identification.21,29 All process steps
and all process parameters in the cell culture process were iden-
tified by cause-and-effect diagram. Part of this cause-and-effect
diagram is shown in Figure 1, indicating that the inoculum
train step has 11 parameters, the production medium reconsti-
tution step has 10 parameters, the feed medium reconstitution
step has 12 parameters, and the production culture step has 16
parameters. In total, the cell culture process was found to have
14 steps and 176 process parameters (data not shown).
Risk analysis and evaluation should be the qualitative or
quantitative process of linking the likelihood of occurrence and
severity of harm and the ability to detect the harm.5 Although
many risk analysis tools are known, and no one tool or set
of tools is applicable to every situation, FMEA is one of the
most common and useful tools for risk analysis in pharmaceu-
tical process development.12,13,16 The risks of all of the process
parameters in the 14 cell culture steps were analyzed and eval-
uated using FMEA. Each process parameter was assessed in
terms of three factors: severity of impact, probability of occur-
rence, and likelihood of detection (Table 1). Severity of impact
was ranked on a five-level scale with a score of 1–11; a score
of 11 indicates that the parameter has already been found to
have a deleterious impact on CQAs or key performance indica-
tors. Probability of occurrence was ranked on a four-level scale

Table 1. Definitions Used for FMEA

Score Definition

(A)
1 It is known that the parameter does not affect CQAs and KPIs because we already have data.
2 The parameter is not expected to affect CQAs and KPIs based on experience and prior knowledge (Other cell lines’ data show no effect).
4 The parameter might affect CQAs and KPIs based on experience, prior knowledge, and literature (Some other cell lines’ data show
effects).
7 The parameter is expected to affect CQAs and KPIs based on experience and prior knowledge (Other cell lines’ data show effects).
11 It is known that the parameter affects CQAs and KPIs because we already have data.
(B)
1 Never in the foreseeable future.
2 Never, but might happen.
3 Rare (less than once per 20 batches, below 5%)
5 Sometimes (more than once per 20 batches, over 5%)
(C)
1 Change can be detected and dealt with immediately.
3 Change can be detected and dealt with within a day.
5 Change cannot be detected or dealt with.

(A) Severity of impact, (B) probability of occurrence, and (C) likelihood of detection.
CQA, critical quality attribute; KPI, key performance indicator.

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 RESEARCH ARTICLE – Pharmaceutical Biotechnology 4277

Table 2. Results of Failure Mode and Effects Analysis for the Production Culture Step

Process Parameter Control Space Characterization Range S O D RPN

IVCD (×105 cells/mL) 2.03–2.79 1.20–2.80 7 2 5 70

Duration (day) 13.4–13.8 13.0–14.0 7 2 5 70
pH (day 4–14) 6.70–6.88 6.65–6.95 7 2 3 42
Feed start timing (h) 72.00–72.02 60.00–96.00 4 2 5 40
pH control start timing (h) 72.00–72.32 60.00–96.00 4 2 5 40
Cell age (day) – 130 7 1 5 35
pH (upper, day 0–3) 7.16–7.28 <7.40 4 2 3 24
Minimum glucose concentration (g/L) – >0.2 4 2 3 24
Feed rate (%) 24.6–28.6 20.0–40.0 4 2 3 24
Dilution rate (%) 7.2–8.1 6.0–9.0 2 2 5 20
Inner pressure (MPa) 0.013–0.017 0.010–0.020 2 2 5 20
Temperature (◦ C) 36.7–37.2 36.5–37.5 7 2 1 14
Agitation power (rpm) 18.0–18.1 17.0–19.0 2 2 3 12
Viability (%) 98.8–99.8 >80.0 4 2 1 8
Working volume (%) −0.4–0.1 −10.0–10.0 2 2 1 4
Dissolved oxygen (%) 28.8–70.9 >10.0 2 2 1 4

IVCD, initial viable cell density; S, severity of impact score; O, probability of occurrence score; D, likelihood of detection score; RPN, risk priority number.

with a score of 1–5; a score of 5 indicates that the parame-
ter has the highest probability of deviating from the control
space. Likelihood of detection was ranked on a three-level scale
with a score of 1–5; a score of 5 indicates that the parameter
cannot be detected or cannot be dealt with even if detected.
Considering that the manufacturing process is operated within
a certain control space, characterization ranges should be wider
than the control space. Characterization ranges were defined
as “ coefficient × control space” and were used throughout the
risk assessment and process characterization as ranges within
which the set point values of process parameters were changed.
A coefficient of 3 was basically used to calculate the character-
ization ranges; however, when the resulting ranges were so
wide that product quality or process performance had to devi-
ate beyond preferable ranges, a coefficient of less than 3 was
used. In the risk assessment, the severity of impact for each
process parameter was evaluated within the characterization
range, whereas probability of occurrence and likelihood of de-
tection for each process parameter were evaluated at the edges
of the control spaces. The risk analysis was conducted on the
basis of information taken from the literature, experimental
and manufacturing data on this and other similar processes
during developments, and general scientific principles.
Table 2 summarizes the risk analysis results for the produc-
tion culture step. Although in the risk analysis, the severity
of impact scores of process parameters were defined with re-
spect to each quality attribute and performance indicator, the
highest score is taken as the severity of impact score. Each
risk priority number (RPN) was calculated by multiplying the
scores for severity of impact, probability of occurrence, and like-
lihood of detection, and the resulting scores were used to judge
which process parameter should be subsequently evaluated in
the process characterization. The RPN threshold was deter-
mined by experts in each function and, based on an acceptable
level of risk at this stage, was set at 40. IVCD, culture duration,
and pH, each of which had an RPN of over 40, were defined as
potential CPPs and were evaluated in the process characteri-
zation. From among all the process steps, FMEA identified no
potential CPPs other than those for the production culture step
(data not shown).
Parameters, which had an RPN below the threshold, were
carefully reviewed by the risk assessment team. Although tem-
perature in the production culture step was not defined as a
potential CPP by FMEA, this parameter was evaluated in the
process characterization for two reasons. One reason was that
the control space and the characterization range for tempera-
ture were very narrow because temperature in the large-scale
bioreactor had been strictly controlled and the risk posed by
variations in temperature might have been undervalued in the
FMEA. The other reason was the concern that there are poten-
tial interaction effects between temperature and other process
parameters. For these two reasons, temperature was also de-
fined as a potential CPP. Material lot—medium lot and other
materials lot in medium reconstitution step—was also defined
independently of the FMEA as a factor that should be evalu-
ated in the production culture step because lot-to-lot variation
in raw material has the potential risk of leading to variability in
quality attributes and performance indicators. Material lot for
the production culture was evaluated to statistically analyze
its variability. Material lot for the subculture was not included
because the purpose of that step is only to passage and expand
cells for production culture and the impact on quality attributes
and performance indicators was considered to be limited. In to-
tal, four process parameters and material lot were evaluated in
process characterization.
Process Characterization
The risk assessment stage of our QbD case study identified
four potential CPPs (IVCD, culture duration, pH, and tem-
perature) and material lot in the production culture step for
experimental evaluation in the process characterization stage.
The actual impact of these potential CPPs and material lot
on quality attributes and performance indicators was evalu-
ated by using a DoE-based multivariate study. Because the
four continuous factors (temperature, pH, IVCD, and duration)
and the one nominal factor (material lot) cannot be comprehen-
sively evaluated by a single experimental design, a three-level
face-centered central composite design to evaluate IVCD, pH,
and temperature was integrated with a two-level full factorial
design to evaluate culture duration and material lot to create

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4278 RESEARCH ARTICLE – Pharmaceutical Biotechnology
Figure 2. Multivariate regression analysis for main peak in CEX. (a) Distribution of main peaks in CEX. (b) Correlation between actual values
and predicted values from the multivariate regression model. (c) Residual analysis for the multivariate regression model. (d) Significant factors
affecting main peak in CEX. IVCD, initial viable cell density.
a single multivariate experimental design. All of the main ef-
fects of the five experimental factors and all of the interaction
effects between the factors could be estimated independently in
this experimental design. Because the ranges evaluated during
process characterization are typically wider than the operating
ranges encountered during manufacturing, we used the char-
acterization ranges as the experimental ranges during process
characterization (The characterization range of temperature
was reset as 36.0–38.0). A total of 30 fed-batch production cul-
ture runs were conducted using 1 L bioreactors, and the titer,
main peak in CEX, H2 L2 in CE-SDS, and high-mannose were
used as potential key performance indicators or potential CQAs
to statistically analyze the impact of process parameters. The
acceptance criteria for the main peak in CEX and for H2 L2 in
CE-SDS were set using the specifications for manufacturing,
and the acceptance criterion for high-mannose was set using
the mean + 3SD of data obtained during process development.
The acceptance criterion for titer was not set because titer does
not affect the efficacy or safety of the mAb in patients.
As a representative example of the procedure used, the
statistical analysis using JMP software (SAS Institute, Cary,
North Carolina) for the main peak in CEX is described in detail
below, although the same analysis was performed for each qual-
ity attribute and performance indicator. First, the distribution
of CEX main peaks was monitored by histogram to confirm that
the data were normally distributed (Fig. 2a). In cases where the
data were not normally distributed (e.g., viability), a variable
Nagashima et al., JOURNAL OF PHARMACEUTICAL SCIENCES 102:4274–4283, 2013
transformation such as logit transformation or Box–Cox trans-
formation was applied before performing statistical analysis
that assumes the analyzed data are normally distributed. The
main effects, interaction effects, and quadratic effects with an
F ratio of over 2.0 were considered to be significant and were
incorporated into a multivariate regression model. The actual
values and the predicted values by the model were shown in a
scatter plot (Fig. 2b).
As an indication of how well the mathematical model fitted
the experimental data, rather than using R2 , which increases
mechanically as more factors are incorporated into the model,
we used adjusted R2 that takes into consideration the number
of factors incorporated. A lack of fit is also a useful indication to
confirm whether sufficient factors including interaction effects
and quadratic effects are incorporated into the model or not.
The error measured for these exact replicates is called pure
error and is the portion of the sample error that cannot be ex-
plained or predicted by the mathematical model. A lack of fit
can be significantly greater than pure error if sufficient factors
including interaction effects and quadratic effects are not in-
corporated into the model. The adjusted R2 and the P value of
lack of fit of the mathematical model were 0.914 and 0.302, re-
spectively, indicating the appropriateness of the mathematical
model. Residuals of mathematical models should be randomly
distributed around zero; a scatter plot of residuals by predicted
values indicated that no notable tendency can be observed
(Fig. 2c). The statistical analysis showed that the main peak
DOI 10.1002/jps.23744

Table 3. Design Space for the Production Culture Step
Potential CPP Range
IVCD (×105 cells/mL) 1.2–2.8
pH 6.65–6.90
Culture duration (day) 13–14
Temperature (◦ C) 36–38
The range of pH in the design space is narrower than the characterization
range because of the deviation of high-mannose from the desired criterion.
CPP, critical process parameter; IVCD, initial viable cell density.
in CEX was most significantly influenced by the main effects of
temperature, pH, IVCD, and culture duration; by the interac-
tion effects between temperature and pH and between temper-
ature and duration; and by the quadratic effects of temperature
and pH (Fig. 2d). Similar results were obtained for the other
responses (data not shown), and the prediction models were
used for the further analysis.
The four statistical prediction models (for main peak in CEX,
titer, H2 L2 in CE-SDS, and high-mannose) were then depicted
as contour plots to clarify the multivariate acceptable range in
the production culture step (Fig. 3). In this case study, spec-
ifications or mean + 3SD from the process development data
were used as acceptance criteria. Deviation beyond the accep-
tance criteria is shown in the contour plots as shaded areas.
Although no deviation was observed for the main peak in CEX
and H2 L2 in CE-SDS, high-mannose levels were higher than
the acceptable criteria in some ranges. These results indicate
that the ranges of some parameters should be narrower than
the characterization range to establish an appropriate design
space for the production culture step. One possible constraint
is to narrow the range of pH from 6.65–6.95 to 6.65–6.90 be-
Figure 3. Understanding the design space in terms of contour plots. Internal x- and y-axes are temperature and pH, respectively. External
x- and y-axes are IVCD and culture duration, respectively. Deviation beyond the acceptance criteria is shown as shaded areas. The dashed line
shows an example of a constraint to establish a design space. IVCD, initial viable cell density.
DOI 10.1002/jps.23744
RESEARCH ARTICLE – Pharmaceutical Biotechnology 4279
cause pH had the biggest impact on high-mannose and this
constraint could completely remove all shaded areas from the
contour plots. Table 3 shows an example design space for the
production culture step in the cell culture process.
Another description of a design space can be achieved by ap-
plying constraints to the prediction models, as has been shown
in other case studies.12,13,16,18,20,21 In such a case, the area within
the criteria with values that predict high-mannose can be de-
fined as the design space. Although this design space will be
wider than the design space shown in Table 3, the description
is complicated and difficult to understand intuitively. If con-
straints are not critical for the process, such as in the current
case study, a simpler description of the design space might be
better.
The distributions of model outputs with random variation in
the parameters and model noise were evaluated by Monte Carlo
simulation. The set point value and the perturbative range
observed in the large-scale production were used as the mean
and SD of each parameter, respectively. The relative sums of
mean error of the models were used as the SD of response, and
a total of 5000 runs were evaluated in the simulation. Figure 4a
shows the statistical parameters used for the simulation, and
the results of the simulations are shown in Figures 4b–4e as
histograms. The minimum values of main peak in CEX, titer,
and H2 L2 in CE-SDS, and the maximum value of high-mannose
were respectively 79.69%, 94.02%, 87.03%, and 7.30%, which
were all within the acceptable criteria. These results indicate
that the production culture step in the cell culture process was
robust even considering possible noise such as mathematical
model noise and perturbative noise in large-scale production.
To promote better understanding of the effect of raw ma-
terials in the cell culture process, further statistical analysis
was performed. The main effect of material lot and the interac-
tion effects between material lot and the other four parameters
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4280 RESEARCH ARTICLE – Pharmaceutical Biotechnology

Figure 4. Identifying the distribution of model outputs using Monte Carlo simulation. (a) Statistical parameters used for the simulation.
The set point value and perturbative range observed in large-scale production were used as the mean and SD of each parameter, respectively.
The relative sums of mean error of models were used as SD of response. Results of 5000 runs in the simulation for (b) main peak in CEX, (c) titer,
(d) H2 L2 in CE-SDS, and (e) high-mannose. IVCD, initial viable cell density.

Figure 5. Robustness analysis for the variability of material lot using the profiles of interaction between material lot and parameters for main
peak in CEX, titer, H2 L2 in CE-SDS, and high-mannose. Each line in the interaction profile represents each material lot. IVCD, initial viable
cell density.

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Figure 6. Qualification of scaled down model. (a) Mean ± 3SD of viable cell density from 2500 L scale production runs (n = 6) are shown
as dashed lines. Viable cell densities from the scaled down model runs (n = 7) are shown as solid lines. (b) Main peak in CEX, (c) titer, and
(d) high-mannose were compared between the scales. Results are expressed as mean ± 3SD. VCD, viable cell density.
were incorporated into the four statistical prediction models
to evaluate differences in variability of material lot. Figure 5
shows the profiles of interaction between material lot and
parameters for main peak in CEX, titer, H2 L2 in CE-SDS, and
high-mannose. For each parameter, the set point value that
results in the smallest difference in response between the dif-
ferent material lots means that the set point is more robust
with respect to the variability of the material lot as compared
with other set points. The effect of temperature on H2 L2 , pH on
H2 L2 , IVCD on H2 L2 , and IVCD on high-mannose were each af-
fected by material lot. Although a lower temperature is prefer-
able if considering only material lot, the resulting lower titer
and higher high-mannose at lower temperature are not accept-
able in the production culture step. Lower pH was the more
robust set point with respect to material lot but lower pH is
not suitable because titer would decrease significantly and too
much CO2 sparging would be needed to maintain low pH. On
the other hand, higher IVCD is preferable and acceptable be-
cause the production process becomes more robust with respect
to material lot and the product remains within the acceptance
criteria with sufficient titer. Although this robustness analysis
with respect to variability in the material lot was performed
only to confirm the process capability in this case study, this
point of view is expected to be efficacious and the cell culture
process is expected to become more robust if this evaluation is
incorporated into process optimization during development.
Qualification of the Scaled Down Model
The ICH guidance documents say that a design space can be
developed at any scale.4 Ideally, the performance of the scaled
down model should be compared with the manufacturing-scale
operation before process characterization. However, the pro-
cess used in this case study is still under development and
DOI 10.1002/jps.23744
RESEARCH ARTICLE – Pharmaceutical Biotechnology 4281
manufacturing-scale data cannot yet be collected. Because
of the absence of manufacturing-scale data, 1 L bioreactor
data were compared with those of the largest scale bioreac-
tor (2500 L) to evaluate the appropriateness of the scaled down
model. The two one-sided t-test (TOST) approach is the prefer-
able equivalence test to confirm that there is no difference be-
tween means of groups30 ; however, TOST requires a threshold
difference. This threshold difference should be set on the basis
of sufficient knowledge of the process obtained during develop-
ment, and as an appropriate threshold difference could not be
set in this case study, comparison of means ± 3SD of the data
obtained at each scale was used instead of TOST for the qualifi-
cation of the scaled down model. Figure 6 shows the comparison
between the scaled down model and the large-scale production
using the data of VCD, titer, main peak in CEX, and H2 L2
in CE-SDS as representative examples. Most of the chrono-
logical VCD data from the scaled down model fell within the
mean ± 3SD values of the large-scale production runs. A slight
difference could be seen in the data for titer, but mean ± 3SD
overlapped at each scale. There was no difference between the
scales for main peak in CEX and H2 L2 in CE-SDS. No difference
could be seen in any of the quality attributes, although some
performance indicators (e.g., CO2 concentration in culture) had
a statistical difference (data not shown). These results indicate
that the scaled down model is appropriate for use in the process
characterization stage.
CONCLUSIONS
In this case study, we have described the application of QbD
elements to the cell culture process in the production of a mAb,
which culminated in the construction of a design space for the
production culture step. Risk assessment of the cell culture
Nagashima et al., JOURNAL OF PHARMACEUTICAL SCIENCES 102:4274–4283, 2013
4282 RESEARCH ARTICLE – Pharmaceutical Biotechnology
process step enhanced our understanding of the processes and
enabled us to efficiently carry out the process characterization
step. The process characterization step was conducted with a
qualified scaled down model using an integrated DoE, and sta-
tistical analysis was used to evaluate the impact of process
parameters on quality attributes and performance indicators,
resulting in the establishment of design space. The design space
established for potential CPPs—IVCD, culture duration, pH,
and temperature—provided information with which to create a
potential control space for manufacturing, which was the con-
trol space followed by the Monte Carlo simulation. Further-
more, the experimental approach enhanced our understanding
of how lot-to-lot variation in raw material affected product qual-
ities and showed the possibility that controlling process param-
eters can control the variability arising from raw materials.
This case study reinforced the robustness of the cell culture
process, and points to innovative approaches for further devel-
oping the process to enhance product quality while maintaining
patient safety. This was our first step with the QbD approach,
and it has provided us a great deal of knowledge and has alerted
us to future issues. This case study focused only on quality risk
management for the cell culture process, process characteri-
zation of the production culture step, and qualification of the
scaled down model; therefore, further study is needed to con-
struct a design space which is not for only a single process step
but for the overall manufacturing process, including the cell
culture process and the purification process. Other QbD ele-
ments such as CQA identification and control strategies need
to be accomplished to complete the QbD approach for pharma-
ceutical development. As risk assessment based on a published
tool23 is currently being discussed and applied for some mAbs
to identify potential CQAs, other aspects of the QbD approach
will be published in the future as a case study. Although many
challenges with the QbD approach still remain for regulatory
submissions, we are convinced that we can reap the benefits
from this approach.
ACKNOWLEDGMENTS
We thank our colleagues at Chugai Pharmaceutical Company
Ltd.: T. Sawamura for cell culture and M. Kato and S. Tanaka
for carrying out antibody purification, CEX, and CE-SDS; and
our colleagues at Chugai Research Institute for Medical Sci-
ence, Inc.: S. Ishijima and M. Kasuga for glycosylation analysis.
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