This study examined the effects of adapting Chinese hamster ovary (CHO) cells producing recombinant proteins to low culture temperatures. CHO cells producing erythropoietin (EPO) or follicle-stimulating hormone (FSH) were adapted to 32°C through repeated batch cultures. While cell growth gradually improved with adaptation, specific productivities (EPO or FSH production per cell) decreased. Adaptation improved hypothermic cell growth but did not enhance recombinant protein production due to lower specific productivities.

1. Journal of Biotechnology 122 (2006) 463–472
Adaptation of Chinese hamster ovary cells to low culture
temperature: Cell growth and recombinant protein production
Sung Kwan Yoon a,b , Jong Kwang Hong a , Seung Ho Choo b , Ji Yong Song b ,
Hong Woo Park c , Gyun Min Lee a,∗
a Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 371-1 Kusong-Dong, Yusong-Gu,
Daejon 305-701, Republic of Korea
b Life Science R&D, LG Chemical Investment, P.O. Box 61, Yusong-Gu, Science Town, Daejon 305-380, Republic of Korea
c Department of Chemical Engineering, Hanyang University, Seoul 133-791, Republic of Korea
Received 25 April 2005; received in revised form 27 August 2005; accepted 21 September 2005
Abstract
Recombinant Chinese hamster ovary (rCHO) cells producing erythropoietin (EPO) and rCHO cells producing follicle-
stimulating hormone (FSH) showed a significant increase in specific productivity (q) when grown at 32 ◦ C compared to 37 ◦ C.
However, low culture temperature suppressed cell growth, and therefore, did not increase volumetric productivity as much as
q. In an attempt to increase the volumetric productivity through improvement of hypothermic growth, EPO producing rCHO
(CHO-EPO) cells and FSH producing rCHO (CHO-FSH) cells were adapted at 32 ◦ C in a repeated batch mode using spinner
flasks. Cell growth of both CHO-EPO and CHO-FSH gradually improved during adaptation at 32 ◦ C. Specific growth rates of
CHO-EPO and CHO-FSH cells at 32 ◦ C, through adaptation, were increased by 73% and 20%, respectively. During adaptation
at 32 ◦ C, mRNA levels of cold-inducible RNA-binding protein (CIRP) of both rCHO cell lines did not change significantly,
suggesting that CIRP expression may not be the only cause for growth suppression at low culture temperature. Unlike cell
growth, the recombinant protein production of both rCHO cell lines was not increased during adaptation due to decreased spe-
cific productivities. The specific EPO productivity and specific FSH productivity were decreased by 49% and 22%, respectively.
Southern blot analyses showed that the decreased specific productivities were not due to the loss of foreign gene copies. Taken
together, improvement of hypothermic cell growth by adaptation does not appear to be applicable for enhanced recombinant
protein production, since specific productivity decreases during adaptation to the low culture temperature.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Adaptation; CHO cells; Cold-inducible RNA-binding protein (CIRP); Erythropoietin (EPO); Follicle-stimulating hormone (FSH);
Hypothermia
∗ Corresponding author. Tel.: +82 42 869 2618; fax: +82 42 869 2610.
E-mail address: gyunminlee@kaist.ac.kr (G.M. Lee).
0168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2005.09.010

2. 464 S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–472
1. Introduction may be able to alleviate growth suppression of rCHO
cells at low culture temperature through adaptation,
Chinese hamster ovary (CHO) cell culture is becom- and thereby, further increase the yield as well as the
ing increasingly important for the production of human volumetric productivity of recombinant protein at low
recombinant proteins especially in the pharmaceuti- culture temperature.
cal field. Increasing recombinant protein yields, and Previously, we investigated the effect of culture
thereby, reducing production costs is a major biotech- temperature on rCHO cell growth and erythropoietin
nological target. Culture temperature is one of the most (EPO) production and found the beneficial effect of
important parameters to be optimized for increasing lowering culture temperature on EPO production (Yoon
recombinant protein yields. CHO cells, like most mam- et al., 2003). Here, we adapted rCHO cells producing
malian cells, are cultivated at 37 ◦ C to simulate the body EPO to low culture temperature to determine whether
environment. the beneficial effect of low culture temperature on EPO
Lowering culture temperature below 37 ◦ C production can be enhanced through adaptation. We
decreases specific growth rate (µ). It has been also monitored the CIRP content of the cells during
suggested that the induction of cold-inducible RNA- adaptation to low culture temperature. Furthermore, to
binding protein (CIRP) is responsible in part for establish generality of the effect of adaptation to low
the growth suppression at low culture temperature culture temperature on CHO cell growth and recombi-
(Nishiyama et al., 1997). Despite growth suppression, nant protein production, rCHO cells producing follicle-
a number of studies demonstrate the beneficial effects stimulating hormone (FSH) were also adapted to low
of low culture temperature. Recombinant CHO culture temperature.
(rCHO) cell culture at low temperature improves cell
viability and reduces contamination by endogenous
CHO cell proteins for a longer period (Furukawa 2. Materials and methods
and Ohsuye, 1998; Yoon et al., 2003). Moreover,
lowering the culture temperature often increases 2.1. Cell lines and culture media
specific productivity (q), though its effect on q is
variable among rCHO cell lines (Yoon et al., 2004). As The rCHO cells producing human EPO (CHO-
a result, recombinant protein yields can be increased EPO, LGE10-9-27) were used in this study (Yoon
significantly by lowering the culture temperature et al., 2003). They were established by transfection
(Bollati-Fogolin et al., 2005; Fox et al., 2004; Yoon of a vector containing the dihydrofolate reductase
et al., 2003). However, due to the decreased µ, no (dhfr) and human EPO gene into dhfr-deficient CHO
substantial increase in a volumetric productivity, cells (DUKX-B11, ATCC CRL-9096) and subsequent
which is also an important factor considered in process DHFR/methotrexate (MTX)-mediated gene amplifica-
optimization, was obtained by lowering culture tion. The stable rCHO cell line producing EPO was
temperature. Accordingly, to maximize the beneficial selected at 5 ␮M MTX. The rCHO cells producing FSH
effect of low culture temperature, growth suppression (CHO-FSH) were established in a similar manner, but
at low culture temperature needs to be alleviated. selected at 0.32 ␮M MTX.
Adaptation of cells to low culture temperature is The proprietary protein-free media, LG-PF5 con-
probably the simplest approach to alleviate the growth taining 5 ␮M MTX and LG-D/F4 containing 0.32 ␮M
suppression to some extent. When subjected to envi- MTX (LG Life Science, Daejon, Korea), were used
ronmental changes, mammalian cells improve their for suspension cultures of CHO-EPO and CHO-
cell growth through adaptation. A successful case FSH cells, respectively. Prior to the suspension cul-
of adaptation was achieved in hyperosmotic culture tures, rCHO cells were maintained in 125 ml spin-
of hybridoma cells for antibody production. Through ner flasks (Bellco Glass, Vineland, NJ) containing
adaptation to hyperosmolar medium, hybridoma cell the corresponding protein-free medium for several
growth was improved at elevated osmolality, resulting weeks on a magnetic stirrer plate (Bellco Glass) at
in the increased final antibody concentration in hyper- 60 rpm, placed in a 5% CO2 incubator, humidified
osmotic batch culture (Chua et al., 1994). Likewise, we at 37.0 ◦ C.

3. S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–472 465
2.2. Cell culture ity (qEPO ) and specific FSH productivity (qFSH ) were
based on the data collected during the growth phase and
For batch cultures, exponentially growing cells at were evaluated from a plot of EPO and FSH concen-
37.0 ◦ C were inoculated at 2 × 105 cells/ml into spinner trations against the time integral values of the growth
flasks with a working volume of 80 ml. The spinner curve, respectively (Renard et al., 1988). The volumet-
flasks were agitated at 60 rpm on a magnetic stirrer ric productivity was calculated by dividing the product
plate (Bellco Glass) placed in 5% CO2 incubators, and concentration by the culture time.
humidified at either 32 ◦ C or 37 ◦ C.
For adaptation experiments, cells were cultivated 2.5. Southern blot hybridization
in a repeated batch mode. When viable cell con-
centration reached (1.5–2.0) × 106 cells/ml, cells were For Southern blot analysis, chromosomal DNA was
harvested by centrifugation and were re-inoculated extracted from rCHO cells using genomic DNA purifi-
at 2 × 105 cells/ml into the spinner flasks containing cation kit (Promega, Madison, WI). After 2 ␮g of chro-
fresh media. Approximately 0.5 ml of culture sample mosomal DNA was treated with EcoRI at 37 ◦ C for
were taken daily from the spinner flasks. Cell con- 18 h, it was loaded on 1% (w/v) agarose gel for elec-
centration was estimated using a hemacytometer and trophoresis. The separated DNA was characterized by
viable cells were distinguished from dead cells using Southern blot hybridization with the corresponding
the trypan blue dye exclusion method. Culture super- probe. For preparation of the EPO probe, human EPO
natants, after centrifugation, were aliquoted and kept cDNA (579 base pairs) was used as a template. For the
frozen at −70 ◦ C until needed for recombinant protein preparation of FSH probes, human FSH cDNAs (351
assays. base pairs for ␣-unit and 390 base pairs for ␤-unit of
FSH) were used as templates. The probes were then
2.3. EPO and FSH assays radioactively labeled by random primed incorporation
of [␣-32 P]dCTP (Amersham, Amersham, UK). Mem-
The secreted EPO concentration in the medium was brane transfer, prehybridization and hybridization were
quantified by enzyme-linked immunosorbent assay performed using the protocol described (Sambrook and
(ELISA), as described previously (Yoon et al., 2003). Russell, 2001). After hybridization and washing, the
In brief, 96-well plates (Nunc) were coated with mon- blots were exposed to a phosphor screen (Molecular
oclonal mouse anti-human EPO antibody (R&D Sys- Dynamics, Sunnyvale, CA) for 3 h and the band inten-
tems, Minneapolis, MN) and blocked with bovine sity was quantitated using PhosphorImager (Molecular
serum albumin (BSA) and Tween 20. The internal EPO Dynamics).
standard and culture supernatants diluted with blocking
buffer were loaded on wells and treated with poly- 2.6. Reverse transcriptase-polymerase chain
clonal rabbit anti-human EPO antibody (R&D Sys- reaction (RT-PCR) analysis
tems) in diluent solution. The horseradish peroxidase
(HRP) conjugated goat anti-rabbit IgG (ICN, Costa The mRNA levels of CIRP and ␤-actin in the cells
Mesa, CA) was used as an enzyme–antibody conju- were quantified by RT-PCR analysis. For RT-PCR, total
gate. The secreted FSH concentration in the medium RNA from cells was extracted with TRI reagentTM
was quantified using an ELISA kit (IBL, Hamburg, (Sigma) according to the protocol provided by the
Germany), according to the protocol provided by the manufacturer. First-strand cDNA was synthesized from
manufacturer. 5 ␮g of total RNA using the SuperScriptTM synthe-
sis system (Invitrogen, Carlsbad, CA). CIRP mRNA
2.4. Calculation of µ, q and volumetric was amplified using the primer pair 5 -CCATGG-
productivity CATCAGATGAAGGC-3 (NCBI gene no. AY359860,
30–50) and 5 -ATGGAAGGACGATCTGGACG-3
The µ was calculated by plotting the logarithm of (NCBI gene no. AY359860, 561–580) for 26 cycles
viable cell concentration versus culture time during the (94 ◦ C for 30 s, 54 ◦ C for 30 s, 72 ◦ C for 60 s). ␤-
exponential growth phase. The specific EPO productiv- actin mRNA was amplified using the primer pair 5 -

4. 466 S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–472
CCAGTTCGCCATGGATGACG-3 (NCBI gene no.
NM007393, 71–90) and 5 -GCAGCTCAGTAACAG-
TCCGC-3 (NCBI gene no. NM007393, 1209–1228)
for 26 cycles (94 ◦ C for 30 s, 56 ◦ C for 30 s, 72 ◦ C for
90 s). The reaction products were then resolved on a
1% agarose gel. DNA bands were visualized using an
ultraviolet light source after staining with 1 ␮g/ml of
ethidium bromide.
3. Results
3.1. Growth and EPO production of CHO-EPO
cells unadapted to low culture temperature
To characterize cell growth and EPO production at
low culture temperature, CHO-EPO cells, which had
been growing at 37 ◦ C, were cultivated at two different
temperatures, 32 ◦ C and 37 ◦ C.
Fig. 1 shows cell growth and EPO production pro-
files during batch cultures. When subjected to low cul-
ture temperature, cell growth was suppressed (Fig. 1A).
At 32 ◦ C, cells started to grow after a long lag period
(∼ h) and the maximum viable cell concentration
=80
obtained was only 1.9 × 106 cells/ml. On the other
hand, cells at 37.0 ◦ C started to grow exponentially
without a lag period and reached a maximum viable cell
concentration of 4.2 × 106 cells/ml. Despite depressed Fig. 1. Effect of culture temperature on cell growth and EPO pro-
duction of CHO-EPO cells ( : 32 ◦ C, ᭹: 37 ◦ C). (A) Viable cell
cell growth, the maximum EPO concentration at 32 ◦ C concentration, (B) EPO concentration and (C) volumetric EPO pro-
was much higher than that at 37 ◦ C (Fig. 1B). The max- ductivity.
imum EPO concentration at 32 ◦ C was 106.2 ␮g/ml,
which was 1.7-fold higher than that at 37 ◦ C. Extended 3.2. Sequential adaptation of CHO-EPO cells to
culture longevity as well as enhanced qEPO at 32 ◦ C was low culture temperature
responsible for increased maximum EPO concentration
at 32 ◦ C. However, the volumetric EPO productivity To determine whether adaptation of cells to low cul-
was rather decreased at 32 ◦ C because of slowed growth ture temperature can alleviate growth suppression, cells
rate (Fig. 2B). The µ, qEPO and maximum volumetric were cultivated at 32 ◦ C in a repeated batch mode using
EPO productivity at 32 ◦ C and 37 ◦ C were summarized spinner flasks. Cells were also cultivated at 37 ◦ C as a
in Table 1. control.
Table 1
Effect of adaptation to low culture temperature on µ, qEPO and maximum volumetric productivity of CHO-EPO cells
Culture Temperature (◦ C) Specific growth Specific EPO productivity, Maximum volumetric
rate, µ (h−1 ) qEPO (␮g/(106 cells h)) productivity (␮g/(ml h))
Unadapted cells 37 0.027 0.26 0.39
32 0.011 0.39 0.28
Adapted cells 32 0.019 0.20 0.18

5. S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–472 467
Fig. 2. Changes of growth of CHO-EPO cells during adaptation at Fig. 3. Changes of EPO production of CHO-EPO cells during adap-
32 ◦ C ( ) and 37 ◦ C (᭹). (A) Viable cell concentration and (B) µ. tation at 32 ◦ C ( ) and 37 ◦ C (᭹). (A) EPO concentration and (B)
qEPO .
Fig. 2 shows adaptive growth responses of cells to
low culture temperature. When cells were cultivated at
32 ◦ C, cell growth rate appeared to increase during ear- (mean ± standard deviation, n = 10) up to 35 genera-
lier culture passages. On the other hand, cell growth rate tions.
at 37 ◦ C appeared to be constant throughout repeated Fig. 3 shows changes of EPO production profiles
batch cultures (Fig. 2A). To quantitate changes in cell during adaptation to low culture temperature. During
growth rate, µ at each batch culture was calculated and earlier culture passages at 32 ◦ C, decrease in EPO pro-
plotted as a function of generations (Fig. 2B). At 32 ◦ C, duction was apparent despite improved cell growth
µ gradually increased from 0.011 h−1 to 0.019 h−1 for rate (Fig. 3A). At 32 ◦ C, qEPO gradually decreased
the first 15 generations, and thereafter, did not increase from 0.39 ␮g/(106 cells h) to 0.20 ␮g/(106 cells h) for
further. These results imply that growth suppression the first 15 generations, which was even lower than
at low culture temperature can be alleviated by an qEPO at 37 ◦ C. Thereafter, qEPO at 32 ◦ C remained
adaptation. However, the µ at 32 ◦ C was still lower almost constant (Fig. 3B). In contrast, qEPO at 37 ◦ C
than that at 37 ◦ C, even after cells were fully adapted. was almost constant at 0.26 ± 0.02 ␮g/(106 cells h) up
At 37 ◦ C, µ was almost constant at 0.027 ± 0.001 h−1 to 35 generations.

6. 468 S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–472
growth rate achieved by adaptation did not increase
EPO production (Fig. 4B and C). In fact, the adaptation
decreased the maximum EPO concentration and the
maximum volumetric productivity by 59% and 35%,
respectively. The µ, qEPO and maximum volumetric
EPO productivity of adapted cells at 32 ◦ C and 37 ◦ C
were also summarized in Table 1.
3.3. Growth and FSH production of CHO-FSH
cells unadapted to low culture temperature
To accept generality of the effect of adaptation to
low culture temperature on growth and recombinant
protein production of CHO cells, CHO-FSH cells were
also adapted to low culture temperature. First, CHO-
FSH cells, which had been growing at 37 ◦ C, were cul-
tivated at two different temperatures, 32 ◦ C and 37 ◦ C
to characterize cell growth and FSH production at low
culture temperature.
Like CHO-EPO cells, growth of CHO-FSH cells
was also suppressed at 32 ◦ C, but to a lesser extent.
No significant lag period was observed and the max-
imum viable cell concentration achieved at 32 ◦ C
(4.7 × 106 cells/ml) was comparable to that at 37 ◦ C
(4.9 × 106 cells/ml). Despite depressed cell growth, the
maximum FSH concentration at 32 ◦ C (7.7 ␮g/ml) was
Fig. 4. Effect of adaptation of cell growth and EPO production of 4.2-fold higher than that at 37 ◦ C (1.8 ␮g/ml) because
CHO-EPO cells at 32 ◦ C (adapted cells: , unadapted cells: ). (A) of both extended culture longevity and enhanced qFSH
Viable cell concentration, (B) EPO concentration and (C) volumetric at 32 ◦ C. Moreover, unlike CHO-EPO cells, the vol-
EPO productivity. umetric FSH productivity was also increased signifi-
cantly at 32 ◦ C, but not as much as the maximum FSH
To evaluate the effect of adaptation to low culture concentration. The µ, qFSH and maximum volumetric
temperature on cell growth and EPO production, a FSH productivity at 32 ◦ C and 37 ◦ C were summarized
batch culture of adapted cells (11 culture passages at in Table 2.
32 ◦ C) was carried out at 32 ◦ C. Fig. 4 shows the pro-
files of cell growth and EPO production of adapted cells 3.4. Sequential adaptation of CHO-FSH cells to
as well as unadapted cells used as a reference (Fig. 1). low culture temperature
Adaptation to low culture temperature significantly
reduced a lag period (Fig. 4A). The µ also increased Like CHO-EPO cells, CHO-FSH cells were culti-
from 0.011 h−1 to 0.019 h−1 . However, improved cell vated at 32 ◦ C and 37 ◦ C in a repeated batch mode.
Table 2
Effect of adaptation to low culture temperature on µ, qFSH and maximum volumetric productivity of CHO-FSH cells
Culture Temperature (◦ C) Specific growth Specific FSH productivity, Maximum volumetric
rate, µ (h−1 ) qFSH (ng/(106 cells h)) productivity (ng/(ml h))
Unadapted cells 37 0.026 8.2 10.3
32 0.015 21.0 27.7
Adapted cells 32 0.018 16.3 17.9

7. S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–472 469
At 37 ◦ C, cell growth rate appeared to be constant perature did not increase FSH production. In fact, the
throughout repeated batch cultures. On the other hand, adaptation decreased the maximum FSH concentration
cell growth rate at 32 ◦ C appeared to slightly increase and the maximum volumetric productivity by 49% and
during subsequent culture passages. Average µ of the 35%, respectively. The µ, qFSH and maximum volu-
first three batches was 0.145 ± 0.001 h−1 , while that of metric FSH productivity of adapted cells at 32 ◦ C and
the last three batches was 0.177 ± 0.001 h−1 . Unlike 37 ◦ C were also summarized in Table 2.
EPO production, a significant decrease in FSH produc-
tion was not observed during repeated batch cultures 3.5. Analysis of CIRP mRNA by RT-PCR
at 32 ◦ C because qFSH was not decreased as much
as qEPO . At 32 ◦ C, qFSH gradually decreased from To investigate whether the alleviation of growth sup-
21.0 ng/(106 cells h) to 16.1 ng/(106 cells h) for the first pression at low culture temperature by adaptation was
15 generations, which was still significantly higher related to down-regulation of CIRP, mRNA contents of
than qFSH at 37 ◦ C. Thereafter, qFSH at 32 ◦ C remained CIPR at different generations of CHO-EPO and CHO-
almost constant. At 37 ◦ C, qFSH was slightly decreased FSH cells were quantified by RT-PCR. The mRNA
from 8.2 ng/(106 cells h) to 6.0 ng/(106 cells h) during content of ␤-actin was used as an internal control for
repeated batch cultures. normalizing mRNA level of CIRP.
As observed in CHO-EPO cells, improved cell Fig. 5 shows the mRNA content of CIRP at differ-
growth rate achieved by adaptation to low culture tem- ent generations. Regardless of cell lines used, the level
Fig. 5. RT-PCR analysis of CIRP in CHO-EPO cells (A and B) and CHO-FSH cells (C and D) at different generations. The RT-PCR of ␤-actin
mRNA was conducted for the normalization of CIRP mRNA level. The relative CIRP mRNA content was estimated as a ratio of the CIRP
mRNA content to ␤-actin mRNA content for each cell sample.

8. 470 S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–472
Fig. 6. Southern blot analysis of EPO genes (A and B) and FSH genes (C and D) at different generations. Arrows indicate the positive control
prepared by digesting the plasmids containing EPO or FSH gene with EcoRI. The relative gene copy numbers were estimated as a ratio of the
DNA content at different generations to that at earliest generations (three generations for EPO and nine generations for FSH).
of CIRP mRNA significantly increased at 32 ◦ C. Its extracted from the cells at different generations of
level was over 4-fold higher than that at 37 ◦ C. Since it CHO-EPO and CHO-FSH cells were characterized by
has been suggested that the induction of CIRP is partly Southern blot hybridization. The band intensities of
responsible for growth suppression at low culture tem- samples prepared at different generations were normal-
perature (Nishiyama et al., 1997), it is expected that the ized by that at the earliest generation.
mRNA level of CIRP decreases during the adaptation As shown in Fig. 6, both EPO and FSH gene con-
to low culture temperature. However, it was observed tents were not changed significantly with increasing
that the CIRP mRNA level of both cell lines, despite generations, suggesting that the decreased qEPO during
improved cell growth, did not decrease during adapta- the adaptation was not due to the loss of foreign gene
tion at 32 ◦ C. Thus, improved cell growth at low culture copies.
temperature by adaptation did not appear to be related
to changes in CIRP transcription level.
4. Discussion
3.6. Analysis of foreign gene copies by Southern
blot hybridization Lowering culture temperature, which is easily exe-
cuted in cell culture processes, has become a popular
To investigate whether decreased q during adapta- method for increasing the product titer of rCHO cells in
tion to low temperature was related to changes in for- commercial processes. However, the low culture tem-
eign gene content in CHO cells, chromosomal DNAs perature suppresses cell growth, and therefore it does

9. S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–472 471
not increase volumetric productivity substantially in during adaptation to low culture temperature, suggest-
batch cultures of rCHO cells. Accordingly, improve- ing that CIRP expression may not be the only cause for
ment of hypothermic cell growth rate may further growth suppression at low culture temperature.
extend the beneficial effect of low culture temperature Unfortunately, the recombinant protein production
to include enhanced volumetric productivity. of both cell lines was not increased during adapta-
At the low culture temperature (32 ◦ C), growth of tion because of decreased qs. The qEPO and qFSH were
both CHO-EPO and CHO-FSH cells used in this study decreased by 49% and 22%, respectively, by adapta-
was significantly depressed, but to a different degree. tion. The q in CHO-FSH cells is more than 10-fold
Despite depressed cell growth, the maximum EPO and lower than in CHO-EPO cells, which may account
FSH concentrations obtained at 32 ◦ C were 1.7-fold for the higher relative productivities in adapted cells
and 4.2-fold higher than those at 37 ◦ C, respectively. producing FSH when compared to those producing
However, because of slowed growth rates, the volu- EPO. Thus, adaptation to the low culture tempera-
metric productivities were not increased as much as the ture increased neither the volumetric productivities
maximum product concentrations. In fact, the volumet- nor the maximum product concentrations of both cell
ric EPO productivity was rather decreased, compared lines because the detrimental effect of decreased q out-
with that at 37 ◦ C. weighed the beneficial effect of increased µ.
When cells are continuously exposed to unfavor- For rCHO cells producing a chimeric antibody,
able growth conditions, they often change the levels of decrease in specific antibody productivity (qAb ) was
many enzymes and accumulation of metabolites to sur- observed in the presence of MTX with increasing gen-
vive, and therefore, improve their growth (Christie and erations and was found to be related to the loss of
Butler, 1999; Chua et al., 1994). Adaptation of BHK-21 antibody gene copies (Kim et al., 1998). To determine
cells to glutamate-based medium was associated with whether the decrease in q during adaptation was related
changes in the activities of enzymes involved in glu- to changes in foreign gene copies, Southern blot analy-
tamate or glutamine metabolism (Christie and Butler, ses of CHO-EPO and CHO-FSH cells were carried out
1999). Accordingly, the adaptation of rCHO cells to and showed that foreign gene copies of both cell lines
the low culture temperature is likely to improve their did not decrease during adaptation. Thus, the decrease
hypothermic cell growth rates. In this study, we investi- in q of CHO-EPO and CHO-FSH cells during adapta-
gated whether an adaptation of rCHO cells (CHO-EPO tion was not due to the loss of foreign gene copies.
and CHO-FSH) to the low temperature can alleviate The decrease in q of CHO-EPO and CHO-FSH
growth suppression at low culture temperature while cells during adaptation may be related to changes in
maintaining enhanced q. µ. When µ and q values collected during adaptation
As expected, hypothermic cell growth of both CHO- were plotted against each other, µ was inversely pro-
EPO and CHO-FSH cells gradually improved during portional to q (Fig. 7). However, in order to determine
adaptation to the low culture temperature. The µs of
CHO-EPO and CHO-FSH cells at 32 ◦ C were increased
by 73% and 20%, respectively, by adaptation.
Although the molecular mechanisms of slow growth
rate associated with lower culture temperature have not
been completely understood (Glofcheski et al., 1993;
Matz et al., 1996), a discovery of the cold-inducible
RNA-binding protein (CIRP) in mouse fibroblasts by
Nishiyama et al. (1997) suggests that growth suppres-
sion at hypothermic conditions is due to an active
response by the cell rather than to passive thermal
effects. Thus, we inferred that the improved hypother-
mic growth by adaptation might be related to changes
in CIRP expression. However, the CIRP mRNA levels Fig. 7. Relationship between µ and q (CHO-EPO cells: , CHO-
of both rCHO cell lines did not change significantly FSH cells: ). The values were collected during adaptation.

10. 472 S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–472
whether this inverse relationship between µ and q Chua, F.K., Yap, M.G., Oh, S.K., 1994. Hyper-stimulation of mon-
during adaptation to the low culture temperature is a oclonal antibody production by high osmolarity stress in eRDF
common phenomenon, more rCHO cell lines need to medium. J. Biotechnol. 37, 265–275.
Christie, A., Butler, M., 1999. The adaptation of BHK cells to a
be tested. Recently, the relationships between µ and non-ammoniagenic glutamate-based culture medium. Biotech-
q were reviewed and discussed in depth (Dinnis and nol. Bioeng. 64, 298–309.
James, 2005). Dinnis, D.M., James, D.C., 2005. Engineering mammalian cell facto-
Improvement of hypothermic cell growth by adapta- ries for improved recombinant monoclonal antibody production:
tion does not appear to be a good strategy for increasing lessons from nature? Biotechnol. Bioeng. 91, 180–189.
Fox, S.R., Patel, U.A., Yap, M.G., Wang, D.I., 2004. Maximizing
volumetric productivity because of decreased q. Alter- interferon-gamma␥ production by Chinese hamster ovary cells
natively, a biphasic process, wherein cells are first cul- through temperature shift optimization: experimental and mod-
tivated at 37 ◦ C in the growth phase for high µ and then eling. Biotechnol. Bioeng. 85, 177–184.
temperature is shifted to low culture temperature in the Furukawa, K., Ohsuye, K., 1998. Effect of culture temperature on a
recombinant CHO cell line producing a C-terminal ␣-amidating
production phase for high q, may be used in order to
enzyme. Cytotechnology 26, 153–164.
increase the volumetric productivity (Bollati-Fogolin Glofcheski, D.J., Borrelli, M.J., Stafford, M., Kruuv, J., 1993. Induc-
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