Biotechnology and Bioprocess Engineering 19: 1097-1104 (2014)
DOI 10.1007/s12257-014-0865-z
RESEARCH PAPER
Production of Recombinant Human Growth Hormone by rCHO
Cells in a Depth Filter Perfusion System
Jae Choon Kim, Hyeong Sun Kwon, and Duk Jae Oh
Received: 13 October 2014 / Revised: 18 December 2014 / Accepted: 20 December 2014
© The Korean Society for Biotechnology and Bioengineering and Springer 2014
Abstract Human growth hormone is a single-chain
polypeptide produced commercially from recombinant animal
cells as well as recombinant microorganisms. Its increased
applications have requested development of highly efficient
production systems using particularly animal cells. Depth
filter perfusion system (DFPS) has been developed and
successfully used for production of recombinant proteins
such as antibodies from recombinant Chinese hamster ovary
(rCHO) cells. In this study, rCHO cells expressing recombi-
nant human growth hormone (rhGH) were successfully
cultivated in the DFPS for 2,200 h. Parameters affecting
the performance of the DFPS for rhGH production were
investgated. The depth filter with 40 µm pores was selected
for stable operation. The shifts of culture temperature and
pH were tested at 37 and 33°C, and 7.2 and 7.0, respectively.
In the begining of the culture, more than 80% of the seeded
cells were immobilized in 200 min by medium/cell circulation.
When the culture temperature was lowered from 37 to
33°C, about 50% increase of the volumetric productivity
(VP) at a perfusion rate of 2.0/day was achieved, and
the VP reached 168 mg/L/d at perfusion rate of 3.0/day,
showing the benefit of cultures in low temperature. In
contrast, when pH was shifted from 7.2 to 7.0, maintaining
rhGH concentration at about 60 mg/L and decreased
perfusion rate from 2.0 to 1.0/day, the VP dropped to 50%.
As the DFPS showed stable and efficient production of
rhGH for long-term periods from this study, it can be an
Jae Choon Kim, Hyeong Sun Kwon, Duk Jae Oh*
Department of Bioscience and Biotechnology, Sejong University, Seoul
143-747, Korea
Tel: +82-2-3408-3764; Fax: +82-2-3409-3764
E-mail: djoh@sejong.ac.kr
Jae Choon Kim
Binex Co., Ltd, Incheon 406-840, Korea
attractive choice for production of rhGH from recombinant
animal cells.
Keywords: human growth hormone, depth filter perfusion
system, Chinese hamster ovary cell, perfusion culture
1. Introduction
Human growth hormone (hGH) is a single-chain polypeptide
that not only promotes growth, but is also responsible for
a variety of metabolic processes [1-3]. The hGH is synthesized
as a 217 amino acid precursor protein, including a terminal
signal peptide removed by proteolytic cleavage during
secretion. The mature, secreted form of the protein is
approximately 191 amino acids in length and has a molecular
mass of about 22 kDa [1,4]. As more rhGH-based drugs
from recombinant animal cells as well as recombinant
microbes are commercialized, increasing demand for a
consistent supply of clinical-grade material at an annual
level of tons is foreseeable. An abundant and safe, albeit
expensive, supply of human growth hormone for human
use can be provided by recombinant DNA and cell culture
technology.
Chinese hamster ovary (CHO) cells are widely used for
the production of therapeutic proteins [5]. To obtain large
amounts of recombinant proteins, it is necessary to combine
a high-density culture of recombinant CHO (rCHO) cells
with optimal environmental parameters. In an effort to
increase recombinant protein productivity in rCHO cells,
the effects of various environmental parameters, such as
temperature, on cell growth and recombinant protein pro-
duction have been extensively investigated. For example,
lowering the culture temperature below 37°C decreases the
specific growth rate, but increases the productivity of
1098
recombinant proteins. Various studies have demonstrated
its beneficial effects [6,7].
Perfusion culture processes can eliminate the nutrient
limitation and inhibitor accumulation problems inherent to
animal cell cultures. As a different approach, perfusion
culture is extensively used in mammalian cell cultures to
increase volumetric protein productivity by achieving
relatively high cell densities, compared with batch and fed-
batch methods [8-11]. Currently, various strategies for
perfusion culture have been reported, including spin filter
[12-14], hollow-fiber [15,16], centrifugal device [17,18],
and dialysis membrane [19,20], to achieve high productivity
through high cell density. However, each perfusion strategy,
although applicable to mammalian cell perfusion culture,
has its respective disadvantages. The main problems of cell
retention devices are membrane fouling and clogging. These
can limit the process duration, destabilize operations, and
make it difficult to sustain a high perfusion rate at high cell
density [21]. There are many advantageous features in the
depth filter perfusion system (DFPS), overcoming the
limitation of conventional perfusion system. First, cells in
DFPS can be readily entrapped in the void space of the
filter matrix by medium circulation and grow within the
fibrous filter matrix free from mechanical shear stresses.
Second, DFPS provides a spacious void matrix for high-
density cell culture, as well as an easy method for stable
long-term operation. Therefore, with those features, the DFPS
could be successfully applied to high-density cultures of
hybridoma, rCHO, and Vero cells [22-26]. In this study, we
demonstrated that the DFPS also could be used for stable
long-term operation for production of recombinant human
growth hormone. We also investigated the effects of culture
parameters (particularly, the shifts of temperature and pH)
on rhGH production from rCHO cells in long-term cultures
using the DFPS.
Fig. 1. Scheme of the depth filter perfusion system (DFPS) consisting of two major compartments: a medium control part (controller +
vessel) and a depth filter part for cell immobilization (depth filter unit).
Biotechnology and Bioprocess Engineering 19: 1097-1104 (2014)
2. Materials and Methods
2.1. Cell line and medium
A recombinant Chinese hamster ovary (rCHO) cell line,
expressing rhGH was used. It was established by co-
transfection of the foreign gene into dhfr- CHO cells
(DG44-origin) followed by subsequent dhfr/methotrexate
(MTX)-mediated gene amplification, and was kindly provided
by PenGen Biotech (Seongnam, Korea). The working clone
of rCHO was selected at 1.00 µM MTX.
Suspension culture of rCHO cells was performed in
a chemically-defined medium, ProCHO5-CDM (LonZa,
Veriers, Belgium), supplemented with 4 mM glutamine and
1 µM MTX.
2.2. Batch cultures in erlenmeyer flasks
Batch suspension cultures were established in 250 mL
Erlenmeyer flasks (Corning Corp., Corning, NY) with an
inoculum of 2.0 × 105 cells/mL in a working volume of
100 mL. A gyratory shaker (n-Biotech, Incheon, Korea) at
110 rpm was used. The Erlenmeyer flasks were incubated
in a humidified 5% CO2 incubator at different temperatures
33 and 37°C. When pH control is required, the pH value of
the culture was measured once or twice a day then adjusted
with 0.1 N HCl or 0.3 N NaOH aqueous solution.
2.3. Depth filter perfusion system
The depth filter perfusion system was introduced and
extensively described in previous studies [22-26]. Briefly,
the overall reactor system consisted of two compartments
as shown in Fig. 1. One was a depth filter matrix part for
cell immobilization (working volume: 0.45 L), and the
other was a reservoir vessel to control medium conditions
(working volume: 0.55 L), connected with BioG-M bioreactor
controller (Biotron Co., Ltd., Bucheon, Korea). The glass
Production of Recombinant Human Growth Hormone by rCHO Cells in a Depth Filter Perfusion System
vessel was equipped with a heating blanket for temperature
control and a marine impeller for gentle mixing. The depth
filter (Profile IITM) was purchased from Pall Corp. (East
Hills, New York, USA) and used as the filter matrix for cell
immobilization. The pore sizes of the filter matrices used in
this experiment were 20 and 40 µm. Depth filters were cut
into 5 cm long, and void volume of 20 and 40 µm filters,
which is the available space where cells can grow, was
measured as about 85 and 95 cm3, respectively. The total
working volume, including a reservoir vessel and the depth
filter matrix, was 1.0 L. The reactor system was normally
operated at 37°C, and then the temperature was shifted
between 37 and 33°C. The pH values were controlled at
7.2 and 7.0 using a 0.3 N NaOH aqueous solution or pure
CO2 gas. When cells were being immobilized, agitation
speed was set to 60 rpm for minimizing shear effects on
cells. After cell immobilization, agitation speed was controlled
to 80 rpm to increase mixing effect in reservoir vessel.
Dissolved oxygen levels were maintained at over 60% by
intermittent sparging of pure oxygen when required. Cells
were inoculated into the reservoir vessel at about 20 ×
105 cells/mL followed by recirculating medium/cell for cell
immobilization.
2.4. Analytical methods
Viable and dead cells were counted directly with a hemacyto-
meter, using the dye exclusion method (0.4% trypan blue in
phosphate-buffered saline). Glucose and lactate concentration
were measured with a YSI 2700 SELECT Biochemistry
Analyzer (YSI Inc., Yellow Springs, Ohio, USA). The rhGH
concentration was determined by indirect enzyme-linked
immunosorbent assay (ELISA). Briefly, Nunc-Immuno plates
(Nunc, Denmark) were coated with the internal rhGH
standard and culture supernatants diluted with coating buffer,
then blocked with blocking buffer (2% bovine serum albumin
in PBS) and treated with goat polyclonal anti-hGH antibody
(R&D Systems, USA). Horseradish peroxidase (HRP)
conjugated with mouse anti-goat IgG antibody (Santa Cruz,
California, USA) was used as an enzyme-antibody conjugate.
Optical density of each well in the plate was measured
at 490 nm using a microplate reader (Vmax, Molecular
Devices, USA) to determine the concentration of the hGH.
3. Results and Discussion
3.1. Immobilization efficiency of the depth filters
Cells in the DFPS can be easily immobilized in the void
space of the filter matrix by medium/cell recirculation and
can grow on/within the fibrous filter matrix. This is one of
the important features of the DFPS that allows this system
stable for long periods even at high cell densities without
1099
Fig. 2. Time courses of immobilization efficiency in depth filters.
More than 80% of the seeded cells were immobilized within
200 min by medium/cells recirculation. (▲) pore size: 20 µm, (□ )
pore size: 40 µm.
filter clogging [23,24]. Immobilization efficiency could be
determined by counting cells in recirculating medium.
Immobilization efficiency (%)
= ⎛1 – --------------------------------------------------------------------------------------⎞ × 100
cell density in recirculating medium
⎝ cell density at inoculation ⎠
As seen in Fig. 2, more than 80% of the seeded cells were
immobilized within 200 min after medium/cell recirculation.
Immobilization efficiency increased rapidly for the first
60 min when using a 20 µm pore size filter, while it increased
gradually for 200 min when using a 40 µm pore size filter,
resulting in similar degrees of immobilization efficiency at
the end. Figs. 3 and 4 show the culture performances in the
perfusion cultures using the different pore size filters, 20
and 40 µm, respectively. In Fig. 3 shows a continuous
perfusion culture in the DFPS using 20 µm pore size filter,
which operated for 1,200 h. It shows stable production of
rhGH in a concentration range of 30 ~ 50 mg/L at the
perfusion rate of 0.5 ~ 2/day. The perfusion rate was
increased stepwise when glucose concentration was below
the value of about 1.5 g/L. The perfusion rate was gradually
increased to support cell growth until 700 h and reached up
to 2/day. In the DFPS, the choice of adequate pore size
filter is important for culture performances to avoid filter
clogging issues as mentioned by Lee et al. [22]. When
20 µm pore size filter was used, after 800 h the filter
clogging started to be observed. The rhGH concentration
suddenly dropped and glucose concentration was gradually
increased and maintained, which meant cells could not
consume glucose properly, representing unsuccessful opera-
tion of the DFPS. The pressure buildup due to filter clogging
made the operation of DFPS difficult. When compared
with previous studies, this filter clogging issue seems to be
1100 Biotechnology and Bioprocess Engineering 19: 1097-1104 (2014)

Fig. 3. Culture profiles from the perfusion culture of rCHO cells

in the DFPS using a 20 µm pore size filter matrix. Temperature,
pH, and DO were controlled at 37°C, 7.2 and 60%, respectively.
( ●) Cell density, ( ■ ) glucose conc., ( □ ) lactate conc., (-)
perfusion rate, and (▲) rhGH conc.
Fig. 4. Culture profiles from the perfusion culture of rCHO cells
in the DFPS using a 40 µm pore size filter matrix. Temperature,
pH, and DO were controlled at 37°C, 7.2 and 60%, respectively.
( ●) Cell density, ( ■ ) glucose conc., ( □ ) lactate conc., (-)
perfusion rate, and (▲) rhGH conc.

cell line and operation process dependent, therefore it should

be investigated and optimized prior to applying to cell
culture process.
When a depth filter with a 40 µm pore size was used
(Fig. 4), production of rhGH in a concentration range of 30
~ 50 mg/L at the perfusion rate of 0.5 ~ 2/day was stably
obtained during 1,200 h culture. For stable operation of the
DFPS, a depth filter with a 40 µm pore size was selected
and used for the experiments followed.
3.2. Effects of temperature and ph on cell growth and
metabolic activities in batch cultures
Prior to investigating the effects of culture temperature and
pH on cell growth, metabolic activities, and rhGH pro-
duction in the DFPS, batch suspension cultures at different
temperature (37 and 33°C) and pH (7.2 and 7.0) were
performed using Erlenmeyer flasks.
As shown in Fig. 5, with about 2-fold increase of final
rhGH concentration, cultivation at lower temperature (33°C)
yielded 2-fold increase in volumetric productivity compared
to that from 37°C. The glucose uptake decreased, while the
lactate production increased in lower temperature. On the
other hand, as shown in Fig. 6, the different values of pH
tested (7.2 and 7.0) did not affect rhGH productivity,
whereas glucose uptake rate and lactate production rate
decreased at pH 7.0 when compared with those from
pH 7.2. At pH 7.2, the maximum cell density and final
rhGH concentration were 33.2 × 105 cells/mL and 64 mg/L,
respectively, and, at pH 7.0, they were 22.1 × 105 cells/mL,
68 mg/L, respectively. The result of batch cultures was
summarized in Table 1.

Table 1. Quantitative summary of batch suspension cultures at different temperature and pH in Erlenmeyer flasks
Temperature (oC) 37 33 37 37
pH Uncontrolled Uncontrolled 7.2 7.0
Maximum viable cell density (105 cells/mL) 26.5 36.3 33.2 22.1
Integral of viable cell density (106 cells/mL/day) 15.3 37.5 36.1 28.0
Final rhGH concentration (mg/L) 68.7 140.2 64.2 67.6
Volumetric productivity (mg//L/day)) 3.12 6.75 3.09 3.25
Glucose uptake rate (g/L/day) 0.29 0.21 0.38 0.33
Lactate production rate (g/L/day) 0.02 0.08 0.08 0.03
Production of Recombinant Human Growth Hormone by rCHO Cells in a Depth Filter Perfusion System
Fig. 5. Batch suspension culture of rCHO cells in Erlenmeyer
flasks at different temperatures (A: 37°C, B; 33°C). ( ○) Viable
cell density, ( ●) Viability, ( ■ ) Glucose conc., ( □ ) Lactate conc.,
( ▲ ) rhGH conc.
3.3. Shifts of culture parameters in long-term perfusion
culture using the DFPS (1. temperature, 2. pH)
The effects of environmental parameters such as temperature
and pH on cell growth and recombinant protein production
in the DFPS were investigated. The medium circulation rate
was initially 100 mL/min, and then increased to 380 mL/min
in order to enhance cell immobilization in the beginning of
the culture. The perfusion rate was increased stepwise,
when glucose concentration decreased to lower than 2 g/L.
Fig. 7 shows a comprehensive overview of the continuous
perfusion culture in the DFPS for 2,200 h using 40 µm
pore size filter.
1101
Fig. 6. Batch suspension culture of rCHO cells in Erlenmeyer
flasks at different pH (A: 7.2, B: 7.0). ( ○) Cell density, ( ●)
Viability, (■ ) Glucose conc., (□ ) Lactate conc., (▲) rhGH conc.
3.3.1. Effects of temperature shift in the DFPS
Temperature is a key environmental parameter that influences
cell growth and recombinant protein production. Most
mammalian cells, including CHO cells, are cultivated at
37°C on the basis of simulating the body environment.
There are many reports showing that lowering culture
temperature below 37°C increases the productivity of
recombinant protein, though the beneficial effects of lowering
the culture temperature on recombinant protein production
appear to depend on cell types and target proteins [6,7,11,27].
As seen in Fig. 7, when temperature was shifted from 37
to 33°C at 365 h, the glucose uptake rate rapidly decreased
from 14.8 to 9.5 g/L/day and lactate production rates also
markedly dropped from 7.62 to 4.74 g/L/day. However, the
1102 Biotechnology and Bioprocess Engineering 19: 1097-1104 (2014)

and VP increased about 40%, compared with the same

perfusion rate (2/day) at 37°C. Under those conditions,
the Y(rhGH/Glu) increased from 10.2 to 13.5 mg/g, showing
effective production of rhGH from consumption of major
energy/carbon source, glucose.

3.3.2. Effects of pH shift in the DFPS

Fig. 7. Perfusion culture of rCHO cells in a DFPS (pore size
40 µm). ( ■ ) Glucose conc., ( □ ) Lactate conc., ( ●) recirculating
cell density, ( ▲ ) rhGH conc., (-) perfusion rate, ( ◆) volumetric
productivity, ( ) glucose uptake rate, GUR, ( ) lactate
production rate, LPR, (★) YrhGH/Glu and (☆) YLac/Glu.
rhGH concentration increased from 56 to 63 mg/L and it
was maintained until culture time of 1,400 h. Table 2
summarizes the effects of the culture temperature in DFPS.
At culture temperature of 33°C, the rhGH concentration
pH is also one of the parameters known to significantly
influence cell culture. Several articles have reported the
effects of pH values on cell culture in a range from pH 6.8
to 7.8. Lowering the culture pH, in general, led to a
significant reduction of the specific growth rate and
glucose consumption, but cell viability remained high for a
prolonged cultivation time, resulting in increased value of
the integral of viable cells at pH levels lower than 7.1 [28-
30]. On the other hand, lowering pH condition sometime
gives rise to negative effects on cell growth and production
yield [30]. In DFPS, after lowering pH from 7.2 to 7.0 at
a culture time of 594 h, the glucose uptake rate decreased
rapidly from 9.5 to 3.9 g/L/day, and then maintained,
which is similar to the results observed from Erlenmeyer
flask cultures at 37°C at different pH values. The rhGH
concentration maintained stably not lower than 67.7 mg/L
after pH shift. However, the VP decreased to 67.7 mg/L/d
from 126.3 mg/L/day, which was associated with lowering
the perfusion rate from 2 to 1/day. When pH was returned
to the original level of 7.2 at culture time of 925 h, we
observed that glucose uptake rate and lactate production
rate were recovered, but VP was not. And the rhGH yield
from glucose consumption, Y(rhGH/Glu) decreased from 17.5
to 11.7 mg/g (Table 2).
When the pH was lowered from 7.2 to 7.0 at a culture
time of 1,452 h again, the glucose uptake rate, rhGH
concentration, and VP decreased gradually. It is very similar
result obtained from our previous works applied to antibody
production [22]. The different response to pH shift in
DFPS and Erlenmeyer flask culture seems to be caused by
the difference between the immobilization system and a
suspension culture system. The gradient of pH value in the

Table 2. Quantitative summary of perfusion culture at different temperature and pH in a DFPS

37oC 33oC 33oC 33oC 33oC
pH 7.2 pH 7.2 pH 7.0 pH 7.2 pH 7.0
Culture time (h) 0 ~ 365 365 ~ 594 594 ~ 925 925 ~ 1,452 1,452 ~ 2,200
Perfusion rate (/day) 0.5 1.0 2.0 3.0 2.0 1.0 1.0 1.0
hGH concentration (mg/L) 51.5 49.1 42.3 56.0 63.2 67.7 65.0 45.7
Volumetric productivity (mg/L/day) 25.8 49.1 84.9 168.0 126.3 67.7 65.0 45.7
Glucose uptake rate (g/L/day) 2.35 4.69 8.27 14.82 9.50 3.91 5.57 3.78
Lactate production rate (g/L/day) 1.18 2.10 4.51 7.62 4.74 2.09 4.16 2.89
Y Lac/Glu (g/g) 0.50 0.45 0.55 0.52 0.50 0.54 0.75 0.76
Y rhGH/Glu (mg/g) 11.0 10.4 10.2 11.5 13.5 17.5 11.7 11.9
Production of Recombinant Human Growth Hormone by rCHO Cells in a Depth Filter Perfusion System
matrix where high density cells are immobilized needs to
be considered prior to setting up the pH value. In DFPS, it
is recommended to set the pH value at 7.2.
4. Conclusion
By investigating immobilization efficiency and cell culture
performace using depth filters of different pore sizes (20
and 40 µm), the optimal pore size of depth filter was
determined to be 40 µm. The DFPS with a 40 µm pore size
filter could be operated without filter clogging, providing
beneficial features such as simplicity, high volumetric
productivity and operational stability. As like our previous
studies using the DFPS that was applied for the cultivation
of hybridoma and rCHO cell for recombinant antibody
producion, the DFPS was proven to be applicable for the
first time to perfusion cultures of rCHO cells expressing
rhGH. Long-term operation of the DFPS was stably per-
formed in association with the shifts of culture temperature
and pH for up to 2,200 h. When the temperature was
lowered from 37 to 33°C, rhGH productivity increased by
50%. The volumetric productivities from the DFPS were
about 20 and 25 times higher than that from batch sus-
pension cultures carried out in Erlenmeyer flasks when the
perfusion rates were 2.0 and 3.0/day, respectively. The pH
shift from 7.2 to 7.0 led to deteriorated performance of the
DFPS, probably caused by pH gradient in cell immobilized
matrix. Thus, pH should be carefully controlled in high cell
density perfusion system, particularly immobilized cell system
such as the DFPS. From this study, it is recommended to
set the pH value at 7.2. From this study applied to rhGH
production, we demonstrated that the DFPS provided its
beneficial features like long-term stable operation up to
2,200 h and high volumetric productivity, appealing again
that DFPS is an attractive choice for production of various
therapeutic biomoleculars using animal cell technology.
Acknowledgement
This work was partly supported by the Technological
innovation R&D program of SMBA [S2063537].
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