Journal of Bioscience and Bioengineering

VOL. xx No. xx, 1e6, 2014

www.elsevier.com/locate/jbiosc

Effect of temperature shift on levels of acidic charge variants in IgG monoclonal

antibodies in Chinese hamster ovary cell culture

Shohei Kishishita,1, 3, * Tomoko Nishikawa,2 Yasuharu Shinoda,2 Hiroaki Nagashima,2 Hiroshi Okamoto,2
Shinya Takuma,2 and Hideki Aoyagi3

Project Planning and Coordination Department, Project and Lifecycle Management Unit, Chugai Pharmaceutical Co., Ltd., 1-1 Nihonbashi-Muromachi 2-Chome, Chuo-ku, Tokyo 103-
8324, Japan,1 API Process Development Department, Pharmaceutical Technology Division, Chugai Pharmaceutical Co., Ltd., 5-1 Ukima 5-Chome, Kita-ku, Tokyo 115-8543, Japan,2 and
Life Science and Bioengineering, Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1 Tennodai 1-Chome, Tsukuba, Ibaraki 305-8572, Japan3

Received 21 August 2014; accepted 30 October 2014

Available online xxx
During the production of therapeutic monoclonal antibodies (mAbs), not only enhancement of mAb productivity but
also control of quality attributes is critical. Charge variants, which are among the most important quality attributes, can
substantially affect the in vitro and in vivo properties of mAbs. During process development for the production of mAbs
in a Chinese hamster ovary cell line, we have observed that an improvement in mAb titer is accompanied by an increase
in the content of acidic charge variants. Here, to help maintain comparability among mAbs, we aimed to identify the
process parameters that controlled the content of acidic charge variants. First, we used a PlacketteBurman design to
identify the effect of selected process parameters on the acidic charge variant content. Eight process parameters were
selected by using a failure modes and effects analysis. Among these, temperature shift was identified from the Plack-
etteBurman design as the factor most influencing the acidic charge variant content. We then investigated in more detail
the effects of shift temperature and temperature shift timing on this content. The content decreased with a shift to a
lower temperature and with earlier timing of this temperature shift. Our observations suggest that PlacketteBurman
designs are advantageous for preliminary screening of bioprocess parameters. We report here for the first time that
temperature downshift is beneficial for effective control of the acidic peak variant content.
Ó 2014, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Acidic charge variants; Chinese hamster ovary cells; IgG monoclonal antibody; PlacketteBurman design; Temperature shift]

Chinese hamster ovary (CHO) cells are popular mammalian
hosts for commercial production of therapeutic proteins (1).
Among these therapeutic molecules, therapeutic monoclonal an-
tibodies (mAbs) are the fastest growing items of interest. They are
now used widely because their high antigenic specificity and low
incidence of unfavorable side effects make them highly effective.
Although mAbs have proven to be useful therapeutic products,
the typical doses of these products required for treatment are
markedly higher than those of most other biologics, resulting in the
need for large-scale production and efficient, cost-effective
manufacturing processes to reduce the cost to patients. In the past
few years, mAb productivity has been enhanced by improving and
refining clone selection, expression vectors, transfection technolo-
gies, and culture media. In the typical optimization of fed-batch
processes, mAb expression levels of 1e5 g/L are currently achieved
(2e4).
Maintaining not only mAb productivity but also mAb quality is
critical, because the mAb molecules produced from CHO cells
contain heterogenous variants that have implications for efficacy
* Corresponding author at: Project Planning and Coordination Department, Proj-
ect and Lifecycle Management Unit, Chugai Pharmaceutical Co., Ltd., 1-1 Nihon-
bashi-Muromachi 2-Chome, Chuo-ku, Tokyo 103-8324, Japan. Tel.: þ81 33 516
5570; fax: þ81 33 281 0217.
E-mail address: kishishitasuh@chugai-pharm.co.jp (S. Kishishita).
and the risk of adverse immune responses in patients. Heteroge-
neity in mAbs is represented by aggregation and the presence of
charge variants, typically as a result of various post-translational
modifications such as oxidation, deamidation, glycosylation, gly-
cation, isomerization, succinimide formation, N-terminal pyroglu-
tamic acid formation, or C-terminal lysine clipping (5e7).
Among the various mAb features that result in heterogeneity,
the content of charge variants is one of the most important, because
charge variants can substantially affect the in vitro and in vivo
properties of mAbs. For example, experiments using chemically
modified mAbs have revealed that charge variants can alter binding
to proteins or cell membrane targets, thus affecting the tissue
penetration, tissue distribution, and pharmacokinetics of the mAbs
(8e15). Therefore, mAb charge variant levels must be controlled
precisely, but there is currently little information available on the
control of these variants by using process parameters; moreover, to
our knowledge there have been no reports on the control of acidic
variant levels.
During process development, we have observed that an
improvement in mAb titer was accompanied by an increase in the
content of acidic charge variants. Here, with the aim of maintaining
comparability among mAbs, we investigated the process parame-
ters affecting the acidic charge variant content of mAbs expressed
in CHO cells. Our strategy was to first adopt a statistical experiment
(PlacketteBurman design) for initial screening of process

1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2014.10.028

Please cite this article in press as: Kishishita, S., et al., Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies
in Chinese hamster ovary cell culture, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.10.028
2 KISHISHITA ET AL. J. BIOSCI. BIOENG.,

parameters affecting the acidic charge variant content. We then TABLE 1. Results of failure mode and effects analysis of the production culture step.
tested in detail the effect of the process parameter selected from Process parameter (unit) Set point Characterization S O D RPN
the screening. (current process) range

IVCD (105 cells/mL) 2.0 1.0e3.0 7 2 5 70

MATERIALS AND METHODS pH () 6.9  0.02 6.8e7.0 7 2 3 42
Temperature shift () Not conducted Not conductede 7 2 3 42
Cell line and cell culture The cell lines used were generated from dihy- 33 C, day 5
drofolate reductase (DHFR)-deficient CHO cells (DXB11, a gift of Dr. L. Chasin) that Feed rate (mL/L) 24.0 12.0e36.0 4 2 5 40
had been adapted to growth in CHO-S-SFM II medium (Life Technologies, Grand Feed timing (day) 3 2e4 4 2 5 40
Island, NY, USA). The expression system used was based on DHFR amplification Initial medium 1.0 0.5e1.5 4 2 3 24
(16,17). The cell lines were transfected with plasmids encoding different IgG mAbs. concentration ()
Cells were passaged regularly in mixed CHO-S-SFM IIeCD-CHO medium (50:50) Feed medium 1000  30 >1000 4 2 3 24
(Life Technologies, Grand Island, NY, USA) supplemented with methotrexate. Cells osmolality (mOsm/kg)
were maintained in suspension in 125-mL Erlenmeyer shake flasks (Corning, NY, DO (%) 40  2 10e60 4 2 3 24
USA) on an orbital shaker (Yamato, Tokyo, Japan) rotating at 100 rpm; the shaker Temperature ( C) 37.0  0.1 36.5e37.5 7 2 1 14
was placed in an incubator at 5% CO2 in air humidified at 37 C. Unless otherwise Cell age (day) e <120 2 1 5 10
specified, the working volume in the 125-mL Erlenmeyer shake flasks was 50 mL. Initial glucose 4.6 4.0e6.0 4 2 1 8
Production culture was performed in fed-batch mode with continuous feeding; concentration (g/L)
model BCC or BCP bioreactors (125 mL or 1 L; ABLE, Tokyo, Japan) were used for Agitation speed 80 60e100 2 2 1 4
production culture. Cells were inoculated at 2.0  105 cells/mL into the of 1-L scale (rpm)
bioreactors. The dissolved oxygen concentration was controlled at 40% of air Working volume () 1.0 0.8e1.2 2 2 1 4
saturation, and the pH was controlled at 7.2 until the end of day 2 and at 6.9 DO, dissolved oxygen; IVCD, initial viable cell density; S, severity of impact score; O,
thereafter by using mixed 1 M NaOHe1 M NaHCO3 solution and CO2. Continuous probability of occurrence score; D, likelihood of detection score; RPN, risk priority
feeding was started on day 3. After production culture, the cell culture fluids were number.
centrifuged (190 g, 5 min) and filtered (0.22 mm; polyethersulfone membrane) to
remove the cells, and antibodies for the analysis of acidic charge variants were
purified from the fluids by using a POROS A20 Protein A column (Life
Technologies, Carlsbad, CA, USA).
probability of occurrence, and likelihood of detection. The RPN
Basal and feed media CHO-S-SFM II e CD-CHO medium was used for pas-
threshold, determined by internal experts, was set at 20. Initial
sage and production culture. Highly concentrated CHO-S-SFM II e CD-CHO medium viable cell density, pH, temperature shift, feed rate, feed timing,
was used as a feed medium. initial medium concentration, osmolality, and dissolved oxygen
Analytical methods Cell counting and cell viability determination were leveldeach of which had an RPN of over 20dwere selected.
performed with a CEDEX model AS20 automated cell counting device (Roche By using a 12-run PlacketteBurman design, we analyzed these
Innovatis, Bielefeld, Germany), and trypan blue exclusion. mAb titers were measured
eight factors for their effects on the content of acidic charge vari-
by protein-A high performance liquid chromatography (Waters, Milford, MA, USA)
assay with appropriate reference standards. We used a POROS A20 Protein A column ants. The factors were tested simultaneously by shifting each of
(Life Technologies). The acidic charge variant content was determined by high- them from a low value (1) to a high value (þ1) (Table 2); among
performance ion-exchange chromatography (Waters). The cation-exchange (CEX) the 12 runs, various combinations of factor levels (1 or þ1) were
column used was a ProPac WCX-10 (Dionex, Sunnyvale, CA, USA). Acidic charge designated according to the PlacketteBurman design (Table 3). One
variants were defined as represented by the several antibody peaks that were eluted
earlier than the main peak during CEX analysis. In contrast, basic charge variants
of the test values (e1 or þ1) was taken as the set point for this
were defined as represented by the several antibody peaks that were eluted later process, whereas the other test value was used as the upper or
than the main peak during CEX. lower limit of the characterization range of FMEA. Regarding se-
Screening of process parameters by PlacketteBurman lection of the upper or lower limit, we chose the limit that we ex-
design PlacketteBurman designs are used to screen the main factors from a pected would have the more positive effect on the mAb titer and
large number of process factors (18). These designs can be very useful in preliminary
quality attributes. For each run, the content of acidic charge variants
studies in which the principal objective is to identify those factors that can be fixed
or excluded in a further optimization process. The variables chosen were initial was measured (Table 3, last column).
viable cell density, pH, temperature shift, feed rate, feed timing, initial medium JMP analysis of the results showed that only temperature shift
concentration, osmolality, and dissolved oxygen. The variables were selected by a had a significant effect on the content of acidic charge variants
failure modes and effects analysis (FMEA) (Table 1). The definitions of the (P < 0.05) (Table 4). Temperature shift was thus a key process factor
assessment factorsdseverity of impact, probability of occurrence, and likelihood
and was selected for further investigation.
of detectiondused for FMEA were developed by some of our study group (19).
Table 2 lists the factors investigated and the values of each factor used in the
experimental design, and Table 3 presents the design matrix. All cell culture was
performed in a 250-mL BCC bioreactor. The duration of cell production culture Effect of shift temperature on acidic charge variant
was 14 days. content Culture was initiated at a density of 2.0  105 cells/mL
Statistical analysis Statistical tests and design of experiments analysis were at 37 C in a 1-L bioreactor. The control conditions were culture at
performed with JMP 9.0 software (SAS Institute, Cary, NC, USA). Where appropriate,
37 C from beginning to end. To determine the effect of temperature
the two-tailed Student’s t-test was performed to determine the statistical signifi-
cance. Statistical significance was defined as P < 0.05. shift on the CHO cell growth, mAb titer, and acidic charge variant
content, on day 5 the temperature was shifted to one of two
RESULTS
TABLE 2. Details of factors selected for the experimental design.
Screening of process parameters by using PlacketteBurman
Factor Units Level
design FMEA is one of the most common and useful tools for risk
analysis in pharmaceutical process development (20e22). We used 1 þ1

FMEA to analyze and evaluate the risks of all of the process pH e 6.8 6.9
parameters for mAb titer and quality attributes in the production IVCD 105 cells/mL 2.0 3.0
Temperature shift 33 C, day5 Not conducted
culture. Each process parameter was assessed in terms of three e
Feed rate mL/h 0.1 0.15
factorsdseverity of impact, probability of occurrence, and Feed timing day 2.0 3.0
likelihood of detectiondon the basis of established scientific Initial medium concentration e 1 1.5
knowledge and our experimental knowledge of mAb titers and Feed medium osmolality mOsm/kg 1000 1200
quality attributes (Table 1). Each risk priority number (RPN) was DO % 40 60

calculated by multiplying the scores for severity of impact, DO, dissolved oxygen. IVCD, initial viable cell density.

Please cite this article in press as: Kishishita, S., et al., Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies
in Chinese hamster ovary cell culture, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.10.028
VOL. xx, 2014 EFFECT OF TEMPERATURE SHIFT ON ACIDIC CHARGE VARIANTS 3

TABLE 3. PlacketteBurman design and resulting contents of acidic charge variants.

No. pH IVCD Temperature shift Feed rate Feed timing Initial medium concentration Osmolality of feed medium DO Acidic charge variants (%)

1 þ1 þ1 þ1 þ1 þ1 þ1 þ1 þ1 33.9
2 1 þ1 1 þ1 þ1 þ1 1 1 25.4
3 1 1 þ1 1 þ1 þ1 þ1 1 30.7
4 þ1 1 1 þ1 1 þ1 þ1 þ1 22.5
5 1 þ1 1 1 þ1 1 þ1 þ1 22.3
6 1 1 þ1 1 1 þ1 1 þ1 32.4
7 1 1 1 þ1 1 1 þ1 1 18.7
8 þ1 1 1 1 þ1 1 1 þ1 21.6
9 þ1 þ1 1 1 1 þ1 1 1 22.1
10 þ1 þ1 þ1 1 1 1 þ1 1 31.6
11 1 þ1 þ1 þ1 1 1 1 þ1 31.7
12 þ1 1 þ1 þ1 þ1 1 1 1 31.7

DO, dissolved oxygen. IVCD, initial viable cell density.

A 180 B 4.0
33°C
150
VCD (105 cells/mL)

35°C

mAb titer (g/L)

3.0 Control
120
*
90 * *
2.0 Shift timing
60 * *
33°C
1.0
30 Shift 35°C
timing Control
0 0.0
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14
Culture time (Days) Culture time (Days)

C
50 *
Acidic charge variant content

*
40
on day 14 (%)

30

20

10

0
33°C 35°C Control

FIG. 1. Effects of different shift temperatures on cell growth, mAb titer, and acidic charge variant content. (A) Viable cell density (VCD). (B) mAb titer. (C) Content of acidic charge
variants on day 14. All data points in panels A to C correspond to the averages of biological triplicates  standard deviation. *P < 0.05 (two-tailed Student’s t-test).

different temperatures, namely 33 C or 35 C. These temperature viable cells than in the control culture. On day 14, the viable cell
shifts were maintained until the end of culture. density in the controls was 65  105 cells/mL. In contrast, on day 14,
We then examined the cell growth profiles (Fig. 1A). From day 10 at 35 C the viable cell concentration was 98  105 cells/mL and at
onward, reducing the temperature maintained greater numbers of 33 C it was 118  105 cells/mL.
We also examined the effect of temperature shift on product
accumulation (Fig. 1B). The maximum mAb titer under control
TABLE 4. Statistical analysis results for PlacketteBurman design. conditions was 3.5 g/L, on day 14. Shifting the temperature to 35 or
33 C resulted in antibody concentrations of 3.1 and 2.8 g/L,
Factors Estimate t-ratio Probability > jtj
respectively. Lowering the culture temperature did not increase
Intercept 27.050 70.08 <0.0001a mAb production.
pH 0.183 0.47 0.6672
IVCD 0.783 2.03 0.1354
We determined the contents of acidic charge variants at the
Temperature shift 4.950 12.82 0.001a different culture temperatures (Fig. 1C). On day 14, the contents of
Feed rate 0.267 0.69 0.5393 acidic charge variants under control conditions and at 35 C and
Feed timing 0.550 1.42 0.2494 33 C were 40%, 30%, and 22%, respectively. There was a significant
Initial medium concentration 0.783 2.03 0.1354
difference in content between the control conditions and a reduced
Osmolality 0.493 1.12 0.3433
DO 0.350 0.91 0.4314 culture temperature of 35 C or 33 C (P < 0.05, two-tailed Student’s
t-test). The lowest level of acidic charge variants was achieved at
DO, dissolved oxygen. IVCD, initial viable cell density.
a
Statistically significant (P < 0.05).
33 C, and an approximately 46% reduction was achieved by

Please cite this article in press as: Kishishita, S., et al., Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies
in Chinese hamster ovary cell culture, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.10.028
4 KISHISHITA ET AL. J. BIOSCI. BIOENG.,

lowering the culture temperature from 37 C to 33 C. Accordingly,
lowering the culture temperature could reduce acidic charge
variant levels, as expected from the results of the PlacketteBurman
design. The content of each charge variant on day 14 is shown in
Fig. S1. The content of main peak was increased by the reduction in
acidic charge variant content.
Effect of timing of temperature shift on acidic charge
variant content Culture was initiated at a density of
2.0  105 cells/mL at 37 C in a 1-L bioreactor. The control culture
was maintained at 37 C until the end of culture. We examined the
effect of temperature shift timing on CHO cells with respect to cell
growth, mAb titer, and acidic charge variant content. Temperature
shifts were made at two different times, namely on day 3 or 5. The
shift temperature was 33 C, and after the shift this temperature
was maintained until the end of culture.
We first examined the viable cell concentration profiles (Fig. 2A).
In the control culture the maximum viable cell concentration was
167  105 cells/mL on day 7. In contrast, the maximum viable cell
concentration with a shift to 33 C on day 5 was 168  105 cells/mL
on day 7, whereas that with a shift on day 3 was 91  105 cells/mL
on day 10. However, although the cells subjected to the early
temperature shift grew more slowly than those subjected to control
conditions or to a later temperature drop, their density plateaued at
day 7 and was still at about the same level at the end of culture.
We then evaluated the effect of the temperature shift timing on
product accumulation (Fig. 2B). The maximum mAb concentration
under control conditions was 3.5 g/L, on day 14. A temperature shift
to 33 C on day 5 gave a maximum mAb concentration of 2.8 g/L, on
day 14, whereas that on day 3 gave an mAb concentration of 1.8 g/L,
on day 14. mAb concentrations were decreased by accelerating the
shift timing.
Next, we examined the changes in acidic charge variant contents
with different timings of the temperature shift to 33 C (Fig. 2C). On
day 14, the acidic charge variant content in the control was 40%. In
contrast, the contents after temperature shifts on days 3 and 5 were
20% and 22%, respectively. Although bringing forward the tem-
perature shift timing from day 5 to day 3 did not further suppress
the acidic charge variant content, in both cases the acidic charge
variant content was markedly lower with the 33 C temperature
shift than in the controls. There was a significant difference in the
content between the control conditions and those in which the
culture temperature was lowered on day 3 or 5 to 33 C (P < 0.05,
two-tailed Student’s t-test). The content of each type of charge
variant on day 14 is shown in Fig. S2. The content of main peak was
increased by the reduction in acidic charge variant content.
DISCUSSION
When process changes are made during mAb productivity
optimization, minimizing changes in quality attributes is critical for
establishing comparability of therapeutic mAbs. During process
development to enhance mAbs productivity, we have sometimes
observed increases in the acidic charge variant content. Our goal
here was therefore to identify the process parameters affecting the
acidic charge variant levels of mAbs expressed in CHO cells.
The first step toward our goal was to select factors (i.e., process
parameters in the production culture) and to assess the risk posed
by each of them to mAb titers and quality attributes. The FMEA
exercise enabled us to eliminate process parameters with low RPN
scores from further study.
We showed here that PlacketteBurman designs could be useful
for minimizing the number of runs required in the initial screening
of numerous potentially influential process parameters. The
resulting data revealed a clear effect of temperature shift on the
acidic charge variant content (Table 4). González-Leal et al. (23)
proposed that PlacketteBurman designs can be used for media
optimization in CHO cell culture. The use of PlacketteBurman

A 200 B 4.0
175 Day3
VCD (105 cells/mL)

Day5
150 Shift 3.0
mAb titer (g/L)

timing Control *
125
100 * 2.0 Shift timing
75 * * * * * *
50 ** Day3 * 1.0
Day5 *
25
Control *
0 0.0
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14
Culture time (Days) Culture time (Days)

C
*
Acidic charge variant content

50
*
40
on day 14 (%)

30

20

10

0
Day 3 Day 5 Control

FIG. 2. Effects of changes in the timing of temperature shift on cell growth, mAb titer, and acidic charge variant content. (A) Viable cell density (VCD). (B) mAb titer. (C) Content of
acidic charge variants on day 14. All data points in panels A to C correspond to the averages of biological triplicates  standard deviation. *P < 0.05 (two-tailed Student’s t-test).

Please cite this article in press as: Kishishita, S., et al., Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies
in Chinese hamster ovary cell culture, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.10.028
VOL. xx, 2014
designs provides a rational approach that could save time and
resources.
Temperature shifts have effects on quality attributes such as
glycosylation (24) and aggregation (25). We found here, for the first
time to our knowledge, that temperature downshifts could also
significantly suppress the levels of acidic charge variants. The acidic
charge variant content was reduced by lowering the temperature
(Fig. 1) and accelerating the temperature shift timing (Fig. 2). A
trend toward reduction in acidic charge variant content by tem-
perature downshift was also observed in another cell line (data not
shown).
These results showed that temperature downshift was a good
way of controlling the acidic charge variant content with the aim of
achieving comparability among mAbs. We obtained the same re-
sults from temperature downshift at a 1000-L bioreactor scale (data
not shown), and we intend to apply this process at the commercial
scale in the near future.
We found here that mAb production was decreased by tem-
perature downshifts. However, many reports have focused on the
beneficial effects of temperature shifts on cell viability and re-
combinant protein production (26e32). The advantages of using
low culture temperatures include maintaining high viability for a
longer culture time and enhancing specific protein productivity.
Therefore, to clarify the wide applicability of the reduction in acidic
charge variant content produced by downshifting the temperature,
we need to determine whether such an effect can be obtained in
cell lines in which productivity is enhanced by temperature shift.
The mechanism by which the content of acidic charge variants is
reduced by a temperature downshift has not yet been elucidated.
Several reports have found that acidic charge variants are generated
by a number of modifications, such as sialic acid (33), glycation (34),
non-reducible species (35), reduced disulfide bonds (35), or dea-
midation in the Fc region (36). To elucidate the mechanism we
intend to try to characterize the kinds of modification that are
affected by the temperature downshift; we will report the results in
the not too distant future.
In conclusion, our results indicate that PlacketteBurman de-
signs are useful for the initial screening of bioprocess parameters
and that a temperature downshift markedly reduces the levels of
acidic peak variants. Further studies are needed to determine
whether temperature shifts are widely applicable to the control of
acidic charge variant levels and to elucidate the mechanism by
which this control occurs.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.jbiosc.2014.10.028.
ACKNOWLEDGMENT
The authors wish to thank all the members of the Chugai
Pharmaceutical team for their support during this work.
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