International Journal of Biotechnology and Biochemistry

ISSN 0973-2691 Volume 6 Number 7 (2010) pp. 1003–1022

© Research India Publications
http://www.ripublication.com/ijbb.htm

Improvement of Recombinant CHO Cell Growth,

Comparison of Growth Characteristics and
Erythropoietin Production

Rambabu Surabattula1*, Muthuvaduganathan Nagasundram2,

Ratnagiri Polavarapu3 and K.R.S. Sambasiva Rao4
1
Scientist, Research & Development, Genomix Molecular Diagnostics Pvt Ltd,
5-36/207, Prashanthnagar, Kukatpally, Hyderabad – 72, AP, India
E-Mail: ramsura@gmail.com
2
Sr. Scientist, Nagarjuna Fertilizers and Chemicals Limited (NFLC), R&D Gowraram
post, Village – warangal, Warangal X road, Medak Dist, Pin – 502279, AP, India
E-Mail: vaduga_2000@yahoo.com
3
President & CEO, Genomix Molecular Diagnostics Pvt Ltd, 5-36/207,
Prashanthnagar, Kukatpally, Hyderabad – 72, AP, India
E-Mail: giripolava@gmail.com
4
Coordinator, Dept. of Biotechnology, Acharya Nagarjuna University, Nagarjuna
Nagar, Guntur, Andhra Pradesh, India
E-Mail: krssrao@yahoo.com

Abstract

Removal of serum from the cell culture media is essential nowadays, since
serum does not have uniformity in composition. The problem with serum free
media is poor growth, serum contains lot of growth nutrients and all are to be
supplemented when it is replaced. We adapted the CHO cells harboring
Erythropoietin gene to serum free media by direct and gradual adaptation
methods. Our observation is that cells grow better in serum free media by
gradual adaptation and it was clearly observed that all commercially available
serum free media doesn’t support the better growth of CHO cells.
Replacement of L-glutamine with glutamax has shown improved growth
(27.5%), protein expression (42%) and decreased ammonia accumulation (3.9
times). Addition of Insulin to serum free medium enhanced CHO cell growth,
protein production and controlled early apoptosis in the batch cultures.
However, DNA fragmentation was seen at 6th day of culture, but it could be
overcome by second addition of Insulin after 48 hrs following the first
addition. Cells were grown in different culture systems by simple batch
process. High cell densities, typically 3.1×106 cells/ml were obtained from
Improvement of Recombinant CHO Cell Growth, Comparison 1004

wave bioreactor with high cell viability and Erythropoietin (EPO) production.

Keywords: Serum free adaptation, Media selection, Glutamine replacement,

Glutamax, Apoptosis, Insulin, Wave bioreactor.

Introduction
Chinese hamster ovary (CHO) cells are of great interest for bio processing and
pharmaceutical research and development[1]. Maintenance of mammalian cell lines
require the addition of serum to the medium, replacement of fetal calf serum is the
most advancement, particularly if it can be achieved by media that are completely
protein-free[2]. Various successful serum free media formulations have been
developed are commercially available today. Unfortunately, many cell lines do not
grow well under serum-free conditions, so it is necessary to determine which serum-
free culture medium can lead to the optimal cell growth. Glutamine, a major nutrient
in mammalian cell culture, is required for the protein and nucleotide synthesis[3].
Glutamine is hydrolyzed metabolically or degrades chemically to ammonia. It has
poor thermal stability, glutamine break down and catabolism result in high ammonia
accumulation. At the end of a recombinant protein production campaign, the
ammonium concentration can reach 5-10 mM[4]. Achievement of high cell yields in
such culture is often limited by an accumulation of ammonia[5]. Cells can grow well
in a medium where glutamine is replaced by glutamate, which is metabolized more
efficiently, reducing the accumulation of ammonia and allows the culture
longevity[6].
Cell death is a major barrier to maintain high cell densities at high viability and
often leads to lower protein yields and quality[7]. Death of mammalian cells[8],
including Chinese hamster ovary cells[9] during cultivation proceeds mainly via
apoptosis. Various strategies such as nutrient feeding, addition of anti-apoptotic
chemicals and genetic manipulation have been used to decrease apoptosis and extend
culture viability[7]. The most common anti-apoptotic agent is insulin, a metabolic
hormone can rescue many types of cells from apoptotic cell death. Anti-apoptotic
effects of insulin are mediated by activation of the phosphatidylinositol-3 kinase
signaling pathway[10].
Dissolved oxygen has significant impact on cell growth, metabolism and product
synthesis[11]. Aeration and agitation variable from vessel to vessel, are the main
factors which directly effect the dissolved oxygen. Hence it is essential to optimize
the vessel conditions for the growth of mammalian cells. Spinner flasks cannot be
used for liquid volumes greater than 1 litre because of insufficient oxygen transfer and
roller bottles are suitable for the adherent cell lines[12]. Bioreactors are complex and
expensive devices, yet they do not provide an ideal environment for cell growth due
to high local fluid shear and bubble aeration. To avoid the problems associated with
cell culture vessels, a new wave bioreactor was designed by Singh[12]. Wave
bioreactors are an excellent choice for the elimination of need of cleaning,
sterilization and associated validation requirements [11]. In this study we describe the
serum free adaptation of CHO cells, selection of commercial serum-free medium from
1005 Rambabu Surabattula et al

the pool of media library, replacement of glutamine by glutamate and glutamax, effect
of insulin addition on cell growth and apoptosis. Studies were also conducted for
selection of culture vessel conditions using simple batch mode to improve the cell
growth and productivity.

Materials and Methods

Cell line and culture media
A cloned recombinant Chinese hamster ovary cell line (CHO-EPO) manipulated to
secrete recombinant human erythropoietin was used in this work. These cells were
kindly provided by Genomix Biotech Inc. (Atlanta, USA). Briefly, they were
established by transfection of a vector containing dihydrofolate reductase (dhfr) and
human EPO genes into dhfr- deficient CHO cells. Media used in this study are
described in Table 1, additives were added in each medium following manufacturer
instructions. All additions to the culture medium were cell culture grade and
purchased from Sigma, Gibco- Invitrogen, if not specified.

Cell culture and maintenance

Several different serum-free media (Table:1) were used to compare their effectiveness
on cell growth and protein expression. Serum free adapted CHO cells were inoculated
at 2×105 cells/ml in to 15 ml of all 7 different types of cell culture media including
control medium (DMEM/F-12 + 5% FBS). All media supplemented with 4 mM
glutamine and 10µg/ml bovine insulin. Cultures were incubated at 37°C and 5% CO2 in
the humidified incubator for 6 days with medium replacement for every 48 hours in all
flasks. In the experiments with glutamine replacement, actively growing CHO cells
(2×105 cells/ml) were transferred in to the culture flasks containing Excell 325 SF
medium with various concentrations (0, 2, 4, 8 and 12 mM) of glutamine, glutamate and
glutamax. Medium was also supplemented with 10µg/ml bovine insulin. Cells were
centrifuged before inoculation to remove the culture medium, particularly to ensure that
remaining glutamine from inoculum preparation was not present.
For Insulin experiments, CHO cells growing at exponential phase were washed
with PBS, and then placed in Excell 325 SF medium containing 4 mM glutamax.
Insulin (10µg/ml) was added in the one flask at 0 hrs (single addition), second flask at
0 and 72 hrs (two additions) and one flask kept for control (without insulin) to see its
effect on cell growth and apoptosis. Glucose (10 mM) and glutamax (4 mM) was
added during the cultivation to avoid their depletion. To determine the growth
characteristics of CHO cells in different culture vessel conditions, cells were grown in
spinner flasks (1000ml, Wheaton), roller bottles (850cm2, Corning) and wave 2-L
perfusion cell bag (GE Health care). All vessels were inoculated at a density of 2×105
cells/ml with 300 ml of Excell 325 medium containing 500 nM MTX, 4mM glutamax
and 10µg/ml insulin (Insulin added at 1st and 3rd d of cultivation). Agitation speeds
were set at 60 rpm (spinner), 20 rpm (roller) and 10 rocks/min (wave bioreactor).
Parallel cultures in T-flask also performed for the comparison of static and agitated
systems. Aeration was kept constant at 0.1 to 0.2lpm for wave cell bag and cultures
were performed at batch mode.
Improvement of Recombinant CHO Cell Growth, Comparison 1006

Analytical Methods
Viable cell concentration and total cell concentration was determined by the trypan
blue exclusion method using a haemocytometer[13]. Secreted EPO concentrations
were quantified by ELISA (EPO.96, MD Bioscience, USA) as for manufacturer’s
instructions. Glucose concentration was determined by enzymatic method (GOP/POD
method, Excel diagnostics, India). Ammonia concentrations were measured using
enzymatic UV method (Randox laboratories, UK).

DNA Fragmentation assay

CHO cell apoptosis was determined by the DNA fragmentation assay on agarose
electrophoresis. Target cell samples were collected in 1.5 ml micro-centrifuge tube,
spun down and resuspended with 0.5 ml 1X PBS, and added 55µl of Triton X-100
lysis buffer (40ml of 0.5M EDTA, 5ml of 1M Tris-HCl buffer (pH 8.0), 5ml of 100%
Triton X-100 and made up to 100ml with MilliQ water), kept for 20 min on ice (4˚C).
Centrifuged the tube in cold at 10000 rpm for 15 minutes, transferred the sample to
new micro-centrifuge tube and then extracted supernatant with 1:1 mixture of phenol:
chloroform and precipitated in two equivalence of cold ethanol and one tenth
equivalence of sodium acetate. The contents were spun down, decanted supernatant
and resuspended the precipitate in 30µl of RNase solution (0.4ml water + 5µl of
50µg/ml RNase) and 5µl of loaded buffer was added. Samples were run on 1.5%
agarose gel, the DNA fragments were observed under Bio-Rad gel doc with UV light
at 254nm and images were captured.

Statistical Analysis
Results are expressed as mean±SD, and the group means are compared with the
Student t test. The accepted level of significance was set at P<0.05.

Results and Discussion

Adaptation to serum free medium
In order to adapt the CHO cells to serum-free cultivation, cells cultured in serum
containing medium were directly sub-cultured into serum-free medium. This direct
adaptation process was performed with initial cell concentration of 2×105 cells/ml.
After growth for 7 days at 37°C, and 5% CO2 with medium replacement every other
day, no viable cells were visible in the flask (Fig:1) and no significant consumption of
glucose was observed. CHO cells are usually grown in media containing fetal bovine
serum (FBS) which contains key components required for cell growth[14]. Serum-
free cultivation of mammalian cells requires many components, which were provided
by serum[15], but these essential nutrients are not present in the DMEM/F-12
medium. It is concluded, this may be the reason for not growing cells in the
DMEM/F-12 medium without serum.
Optimal cell concentration is essential when cells are growing in the serum- free
conditions; Literature recommends a minimum of 2–5×105 cells/ml for adaptation into
a serum-free suspension culture. It was observed that a critical initial cell density was
required for successful growth of chick embryo cells in tissue culture[16-17]. Hence,
1007 Rambabu Surabattula et al

various initial cell concentrations (2, 4 & 6 ×105 cells/ml) were tested (data not
shown) but the same results were obtained. However, commercially available serum-
free media generally include growth factors such as insulin and transferrin,
substituting for mitogenic factors in serum[18]. It is decided to use commercial
serum-free medium (Excell-302) for the direct adaptation, during this adaptation,
small clumps appeared, but cells stop growing after third day of culture (Fig: 2).
During the initial days of culture, cells utilized the glucose and then they entered into
death phase resulting in the decision to stop further evaluation of this procedure.
Cells are conditioned to grow better in serum-free media by a gradual adaptation
procedure [19]. Gradual reduction of the serum concentration increases the chance for
successful adaptation of cells to serum-free environment [20]. For this reason, we
decided to stepwise decrease the serum concentration in every other passage by 50%
to wean cells off serum. The gradual adaptation to serum-free medium gave better
results (Fig: 3). Cells were grown well (Fig: 4A) and viability (Fig: 4B) was
maintained more than 84% during the adaptation. Glucose consumption was observed
during the entire adaptation, as a result of active cell growth there were sharp decline
in glucose concentration. Only a slight reduction in viable cell number occurred when
the serum content was reduced from 5 to 1%. The cell viability rapidly decreased
(Fig: 4B), however, from 1% to 0% FBS, indicating a serum component becoming
growth limiting [21]. Compared to serum containing cultures, reduction of cell growth
is generally observed when cells are transferred to serum-free media [22-24]. In most
cases the proportion of suspended cells increased with decreasing serum
concentration. Therefore, gradual adaptation of CHO cells were considered fully
adapted to the serum-free medium.

Table 1: Cell culture media used.

ID Medium Name
C Gibco- DMEM/F-12 + 5 %FBS

1 Sigma - CHO-DHFR Medium

2 JRH- Ex-Cell 302 SF Medium

3 JRH- Ex-Cell 325 SF & PF Medium
4 Invitrogen- CD CHO Medium
5 Invitrogen-CHO S SFM Medium
6 Cellgro- Cell gro free Medium
7 Hyclone- SFM4CHO Utility Medium
Improvement of Recombinant CHO Cell Growth, Comparison 1008

12 Hrs 48 Hrs 96 Hrs

Figure 1: Growth of recombinant CHO cells in serum-free DMEM/F-12 medium at

different culture hours.

12 Hrs 48 Hrs 120 Hrs

Figure 2: Growth of recombinant CHO cells in serum-free Excell 302 medium at

different culture times.

A B C

D E

Figure 3: Different stages of gradual serum-free adaptation of CHO cells in Excell 302
medium. (A) 5% serum, (B) 2.5% serum, (C) 1% serum, (D) 0.5% serum, and (E) 0%.
1009 Rambabu Surabattula et al

1.6 94

1.4
5%
2.5% A 92 B
1%
0.5%
Viable cells (millions/m l)

1.2 90
0.2%
0%
1.0 88

Viability (%)
0.8 86

0.6 84
5%
0.4 82 2.5%
1%
0.5%
0.2 80
0.2%
0%
0.0 78
0 2 4 6 8 0 2 4 6 8

Culture days Culture days

Figure 4: Growth (A) and viability (B) profile of rCHO cells during the gradual
adaptation to serum-free Excell 302 medium. All values are represented as mean±SD
obtained from three independent experiments.

Serum-free medium (SFM) selection

One of the major problem associated with SFM, is that there is no one universal
medium on which all cells will grow. It has been found that almost every cell line
requires a different set of growth factors, hormones and attachment factors for the
optimal cell growth and yield [15]. Although there is serum or protein-free media
described for the culture of CHO cells [25], they are not really satisfactory, because
they were developed for one special cell clone. In the present work, over seven
different serum-free media from six vendors have been screened to determine the best
medium for growth and productivity.
Cell growth rates were calculated for all the media tested. Cells showed inability
to grow well in all the media (Fig: 5A). When all the media were inoculated with
same no of cells, the highest cell densities were obtained on Excell 325
(1.56±0.036×106) and Excell 302 (1.483±0.016×106 cells/ml) media. By using
Invitrogen CD CHO and CHO S SFM medium, cell densities reached 0.48±0.013×106
and 0.79±0.015×106 cells/ml, whereas cell density level was 0.92±0.025×106 cells/ml
in Sigma medium. 0.58±0.014×106 cells/ml & 0.89 ±0.02×106 cells/ml, cell densities
were observed in Cellgro and Hyclone media. More or less similar growth support
was observed in Excell 325 and Excell 302 media, when compared with control
medium (1.5 ± 0.01×106 cells/ml in DMEM/F-12 + 5% FBS).
CHO cell line demonstrated good viability in both Excell media during the entire
culture period, with viabilities ranging from 87 – 90% (Fig: 5B). Protein production
demonstrated variations in all the media tested during the entire cultivation (Fig: 5D).
Higher consumption of glucose was observed in both Excell 302 and 325 media (Fig:
5C). These results show that Excell 325 and Excell 302 media are the most suitable
for CHO cell growth and protein expression. Large variations were found in both
growth and productivity for the choice of media. While the commonly used serum-
free media no longer contains animal serum as an additive, however they contains
Improvement of Recombinant CHO Cell Growth, Comparison 1010

animal derived components, particularly bovine proteins such as albumin, insulin,

transferrin and lipoproteins. It is highly desirable to develop animal protein-free
media for cell culture processes[26], because any animal derived proteins could carry
a theoretical risk of introducing prions[27] or other adventitious agents[28]. Although
SFM is a step in the right direction it does not entirely alleviate all of the problems
associated with serum, it merely reduces them, this has led to the development of
protein-free media[29-30]. The main benefit of a protein-free media is in the
downstream recovery of product. Hence, it was decided to use only Excell 325
medium (SF & PF) for the further developmental studies.

1.8 92

B
1

A
1.6 2 90
3
1.4 4 88
Viable cells (Millions/ml)

5
1.2 6 86
7
Viability (%)

C
1.0 84

0.8 82 1
2
0.6 80 3
4
0.4 78 5
6
0.2 76 7
C
0.0 74
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7

Culture days Culture days

5 5
Day 2

C D
Day 1
Day 2 Day 4
Day 3 Day 6
4 4
Day 4
Day 5
Day 6
Glucose (mM)

EPO (mg/l)

3 3

2 2

1 1

0 0
1 2 3 4 5 6 7 C 1 2 3 4 5 6 7 C

Media number Media number

Figure 5: Results of the cultivation of rCHO cells in various serum-free media for a
period of 6 days, cell growth (A), viability (B), glucose consumption (C) and EPO
productivity (D). Values reflect the mean and standard deviation of three independent
determinations.

Effect of L-glutamine, glutamate and glutamax on cell growth & protein

production
In figure 6A it can be observed that the 4 mM glutamine gave highest cell growth,
both the growth rate and final cell concentrations were low with the increase in
glutamine concentration (8 and 12 mM). Cells did not grow at all in the medium
without glutamine and cells grown at 2 mM glutamine were slightly higher than the
1011 Rambabu Surabattula et al

higher glutamine concentration. At the end of the incubation, the highest cell density
observed at 4 mM was 1.21±0.04×106; this was almost 33.8-45.5% (Student’s t-test
p<0.001) higher than the other concentrations.
Accumulation of ammonia was significantly high (11±0.24 & 11.9±0.23mM,
Student’s t-test p<0.001) in 8 and 12 mM glutamine, when compared with other
concentrations (Fig: 6C). Cells stop growing at the ammonia concentration higher
than 8 mM. It can be observed that glucose is consumed less (8.8±0.34, 8.2±0.29 &
7.6±0.31 mM) at the lower (2 mM) and higher concentrations (8 & 12 mM) of
glutamine (Fig: 6B), where it was consumed more than 25.4-35.6% (Student’s t-test
p<0.002) higher in the medium containing 4 mM glutamine (11.8±0.31 mM, ‘starting
medium glucose concentration was 20 mM and it was reached to 8.26 mM at the end
of cultivation’). Figure 6D shows EPO concentration in the medium containing
various concentrations of glutamine. The yield of EPO was significantly (3.55±0.02
mg/l, Student’s t-test p<0.001) higher in the culture containing 4 mM glutamine than
other concentrations.

1.4 22

1.2
0 mM
2 mM
4 mM A 20 B
Glucose concentration (mM)

8 mM
Viable cells (Millions/ml)

1.0 18
12 mM

0.8 16

0.6 14

0.4 12

10 0 mM
0.2
2 mM
4 mM
0.0 8 8 mM
12 mM
6
0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 120 140 160 180

Incubation Hours Incubation Hours

14 4
0 mM

D
0 mM

C
12 2 mM
2 mM
4 mM
Ammonia concentration (mM)

3 4 mM
8 mM
10 8 mM
12 mM
12 mM
8
EPO (mg/l)

1
4

2
0
0

0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 120 140 160 180

Incubation Hours Incubation Hours

Figure 6: Comparison of the batch cultures of rCHO cells at different concentrations

of L-glutamine: cell growth (A), glucose consumption (B), ammonia accumulation
(C) and EPO concentration (D). Error bars represent the standard deviations
calculated from the data obtained from three individual experiments.

In the series of experiments for glutamine replacement, same approach was

followed to determine the effect of glutamate on CHO cell growth. Cell densities were
more or less similar (1.45±0.07×106 & 1.39±0.076×106) at concentrations of 4 and 8
mM, on the other hand at 12 mM, it was 1.21±0.027×106 cells/ml which was
Improvement of Recombinant CHO Cell Growth, Comparison 1012

significantly (Student’s t-test p<0.02) lower than the above concentrations (4 & 8
mM) (Fig: 7A). Interestingly, the maximum concentration of ammonia accumulation
was only 3.6±0.08 mM at the highest concentration of glutamate (12 mM) (Fig: 7C);
which was 3 fold less when compared with 12 mM glutamine cultures (11.9±0.23
mM). No ammonia build up was observed in 2 mM glutamate culture. The trends for
the consumption of glucose can clearly be distinguished; it was less (8.1±0.27 mM,
Student’s t-test p<0.001) at the low concentration (2 mM) of glutamate, when
compared with other concentrations (4, 8 &12 mM) (Fig: 7B). Figure 7D shows the
expression of EPO at various concentrations of glutamate, highest EPO production
(4.2±0.08 mg/l, Student’s t-test p<0.002) was observed at the 8 mM glutamate
concentration, but this was insignificant (4.09±0.05 mg/l, Student’s t-test p<0.09)
with the 4 mM glutamate cultures.

1.8 22
0 mM
1.6 2 mM 20

A
4 mM
1.4 8 mM 18
Viable cells (Millions/ml)

12 mM

B
1.2
16
Glucose (mM)

1.0
14
0.8
12
0.6
10
0.4
0 mM
8 2 mM
0.2
4 mM
6 8 mM
0.0
12 mM
4
0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 120 140 160 180

Incubation Hours Incubation Hours

4 5

D
4 mM 0 mM

C
8 mM 2 mM
12 mM 4 4 mM
3 8 mM
12 mM
3
Ammonia (mM)

EPO (mg/l)

1
1

0
0

0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 120 140 160 180

Incubation Hours Incubation Hours

Figure 7: Comparison of batch cultures of the rCHO cells at different concentrations

of glutamate: cell growth (A), glucose consumption (B), ammonia accumulation (C)
and (D) EPO production. Error bars represent the standard deviations calculated from
the data obtained from three individual experiments.

In the experiments with glutamax, maximum cell density was obtained at 4 mM

(1.67±0.022×106, Student’s t-test p<0.049), but this was insignificant (1.59±0.08×106,
Student’s t-test p<0.2) with the 8 mM glutamax cultures (Fig: 8A). However, cells at
highest concentration of glutamax (12 mM) grew with very similar growth profiles to
1013 Rambabu Surabattula et al

4 & 8 mM glutamax. Glucose consumption was significantly (14.8±0.26 mM,

Student’s t-test p<0.029) high with the culture containing 4 mM glutamax (Fig 8B).
Near similar consumption rate was observed in 4, 8 & 12 mM, but in between these
all, high consumption was observed at 4 mM glutamax. Very less accumulation of
ammonia was observed in all concentrations of glutamax used. 3±0.15 mM is the
highest ammonia production observed at 12 mM glutamax cultures (Fig 8C). As seen
in Fig 8D, highest EPO production (6.2±0.2 mg/l, Student’s t-test p<0.021) was
observed with the culture containing 4 mM glutamax at the end of incubation (i.e.,
170 hrs). Where as, 2.7±0.18, 5.9±0.13 & 4.79±0.16 mg/l was obtained in the cultures
with 2, 8 & 12 mM glutamax.
The only prior reports on glutamine replacement with glutamate have been studied
on the human diploid cell lines[31], reported that the replacement of glutamine with
glutamate will increase the cell growth and productivity. Altamirano et al.,[32]
improved the CHO cell culture media formulations by substituting the glutamine with
glutamate. In those studies, they did not clearly state the effect of ammonia on cell
growth and productivity of CHO cell line. In the present work, two different
components were tested (glutamate and glutamax) with the main objective of
obtaining a more efficient use of the nitrogen source, with a lower release of
ammonium ions.

1.8 22
0 mM
1.6 20

A
2 mM
4 mM
1.4 8 mM 18
Viable cells (Millions/ml)

B
12 mM
1.2
16
Glucose (mM)

1.0
14
0.8
12
0.6
10
0.4 0 mM
8 2 mM
0.2 4 mM
8 mM
6
0.0 12 mM

4
0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 120 140 160 180

Incubation Hours Incubation Hours

3.5 7

4 mM 0 mM

C D
3.0 8 mM 6
2 mM
12 mM 4 mM
2.5 5 8 mM
12 mM
Ammonia (mM)

2.0 4
EPO (mg/l)

1.5 3

1.0 2

0.5 1

0.0 0

0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 120 140 160 180

Incubation Hours Incubation Hours

Figure 8: Comparison of batch cultures of the rCHO cells at different concentrations

of glutamax: cell growth (A), glucose consumption (B), ammonia accumulation (C)
and (D) EPO production. Error bars represent the standard deviations calculated from
the data obtained from three individual experiments.
Improvement of Recombinant CHO Cell Growth, Comparison 1014

Toxic levels of ammonia accumulation were crossed at day 4 in 8 & 12 mM

glutamine cultures, cells stop growing in the same culture and the final level of
ammonia production was reached to more than 11 mM in the same flasks (Fig: 6C).
Cells stop growing at the level of 8 mM ammonia in the batch cultivation[33]. Where
as in the experiments with glutamate and glutamax, maximum ammonia
accumulations obtained were only 3.6±0.08 & 3±0.15 mM (Fig: 7C & 8C); this was
too low to produce any inhibition[33]. The accumulation of ammonia was
significantly very low in the media containing glutamine based dipeptide (L-
glutamine and L-alanyl)[34]. However, the cell growth was inhibited at higher
concentration of glutamate (1.21±0.027×106, Student’s t-test p<0.021), there was no
significant growth inhibition was observed at any concentrations of glutamax except 2
mM, this may be due to insufficient level of glutamine.
Cell viability and EPO concentrations were decreased with the increased
accumulation ammonia. EPO content in the cultures grown at glutamate and glutamax
was gradually increased up to the end of cultivation, except the low concentration (2
mM), where it was decreased at the day 5. More than 42% increase in maximum EPO
concentration was achieved by replacing the glutamine with glutamax. Similarly,
15.5% higher EPO concentration was achieved by glutamate. When comparing the
EPO expression in between glutamax and glutamate, there is a significant (32%,
Student’s t-test p<0.001) increase was observed with glutamax than glutamate.
Similarly, cell growth rate was also increased (13.5%, Student’s t-test p<0.007).
Overall, the glutamax increased cell viability, utilized more glucose, produced less
ammonia and yielded more EPO, when compared to glutamine & glutamate.

Effect of Insulin on rCHO cell growth and apoptosis

The effect of insulin addition (10µg/ml) at 0 & 72 hrs on the viable cell concentration
and apoptosis was investigated during the serum-free cultivation. The addition of
insulin at 0 h (1.6±0.017×106/ml, Student’s t-test p<0.001) and 0 & 72 hrs
(1.82±0.016×106/ml, Student’s t-test p<0.001) resulted in a markedly higher viable
cell concentration compared with the control (0.98±0.01×106/ml) (Fig: 9A). While the
viability decreased rapidly to 72±1% from the initial viability of 94% at the end of the
culture without insulin (Fig: 9B). A marked decrease of viability (89±0.5%) was
obtained in the cultures supplemented with insulin only at 0 h. CHO cells are
characteristically begin to lose viability around days 4-5 of culture[35]. A significant
increase of viability (96±0.07%, Student’s t-test p<0.001) was observed in cultures
with double addition of insulin. However, in both cases upto 72 hrs the viability was
super imposable.
In both cases (single & double), insulin addition increased the specific glucose
consumption rate. Consequently, insulin added not only initially, but also during the
period of cultivation increased the viable cell concentration together with the specific
glucose consumption rate (Fig: 9C). It was examined whether insulin addition could
rescue CHO cells from apoptosis by a DNA fragmentation assay. It was found that
DNA from cells treated with insulin (single & double addition) was uncleaved until
3rd day of incubation but the DNA from untreated cells was partially cleaved at the
same day. DNA from the control cells showed ladder formation at the 6th day of
1015 Rambabu Surabattula et al

culture, whereas from insulin double addition it was still uncleaved. DNA from
insulin single addition cells were cleaved partially and showed the ladder formation at
the same day (Fig: 9D). These findings suggest the insulin can retard apoptosis of
CHO cells.
Double addition of insulin apparently increased cell concentration (12% & 46%),
viability (7.5% & 25%) and glucose uptake (26% & 44.7%) compared with single
addition at 0 h and control cultures. The concentration of insulin added at 0 h rapidly
decreased to approximately 0.07% within initial days of cultures. On the other hand,
the insulin concentration was maintained at approximately 0.26% until 144 h after the
2nd addition at 24 h[36]. This may be the reason for not increasing the cell
concentration and viability in cultures with insulin added at 0 h comparatively with
repeated additions. Previous work has shown that withdrawal of insulin lead to a
reduction in viable cell density and viability[37]. The addition of insulin also
increased the production of EPO (Table: 2), this indicates that this effect was
concomitant to the effect on viable cell maintenance (Fig: 9A). Viability loss due to
apoptosis often limits recombinant protein production[7], based on the evidence that
single addition of insulin to the serum-free culture had only a small effect on viable,
total cell concentration and EPO production. The increase in EPO production by the
insulin may be a result of the increased viability and total cell concentration. Insulin is
well known antiapoptotic factor that regulates gene expression in various cells[38-39].
It is expected that the suppression of apoptosis during the production phase in serum-
free or low serum media might increase protein production by mammalian cells such
as CHO cells[40]. The production of EPO was 17.2% & 2.44 fold higher in double
addition of insulin than in single addition and control cultures. Overcoming apoptosis,
the major mode of cell death in many bioprocesses is desirable to enhance product
yield and quality[41].

Table 2: Effect of the insulin addition on EPO production.

Insulin addition EPO (mg/l)

Control (without addition) 3.11 ±0.12
Single addition at 0 h 6.29 ±0.16
Two addition’s at 0 & 72 hrs 7.6 ±0.15
The concentration of EPO at the end of (day 7) culture, shown in Fig: 9, was
determined.
Improvement of Recombinant CHO Cell Growth, Comparison 1016

2.0 100

1.8 Control
Single addition 95
Repeated addition
Viable cell density (cells/ml)

1.6

1.4 90

Viability (%)
1.2
85
1.0
80
0.8

0.6 75

0.4 Control

0.2 A 70 Single addition

Repeated addition B
0.0 65
0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 120 140 160 180

Time (hours) Time (hours)

M 1 2 3 4 5 6
30

28 Control D
26
Single addition
Repeated addition C
24
Glucose (mM)

22

20

18

16

14

12

10
0 20 40 60 80 100 120 140 160 180

Time (hours)

Figure 9: Effect of insulin additions on CHO cells growth (A), viability (B), glucose
consumption (C) and apoptosis (D). Inter-nucleosomal cleavage of CHO cell DNA
during apoptosis (D), Isolated chromosomal DNA (2 µg) was electrophoresed on
1.5% agarose gel and the fragmented DNAs were viewed by staining with ethidium
bromide and ultraviolet transillumination. DNA samples were collected at 3 & 6 day
of cultures. M: a molecular weight marker, 200-bp DNA ladder, lane 1 & 4: DNA
isolated from the cultures treated with double addition of insulin at 0 & 72 hrs, lane 2
& 5: from the cultures treated with single addition of insulin at 0 hrs, lane 3 & 6: from
the cultures treated without insulin. Arrows indicates the addition of insulin (A & B)
and glucose (C) during the cultivation.

Culture vessel comparison for CHO cells grown in agitated systems

In an effort to increase CHO cell growth and protein production, the effect of different
culture vessel conditions on cell growth and protein production in serum-free medium
were investigated Cultures grown for 7 days in wave bioreactor achieved higher cell
densities (3.1±0.1×106 cells/ml) than those grown in spinner flask (1.8±0.03×106
cells/ml), roller bottle (2.22±0.12×106 cells/ml) and T-flask (1.56±0.11×106 cells/ml)
(Fig: 10A). Maximum cell density was obtained at 5th day of culture in wave
bioreactor and viable cells started decrease after the same day, 49.67% (Student’s t-
test p<0.001) higher cell growth was observed in wave bioreactor compared to static
1017 Rambabu Surabattula et al

culture; where as only 13% & 29.7% were seen in spinner and roller bottles,
respectively. Significant decrease in viability (Fig: 10B) was observed with wave
bioreactor after reaching to high cell density, this clearly indicates the depletion of
nutrients in the medium. No significant decrease in viability was obtained in other
cultures.

3.5 97
T-Flask
T-Flask
Spinner Flask

B
3.0 Spinner Flask
96 Roller Bottle
Roller Bottle
Wave Bioreactor
Viable Cells (Millions/ml)

Wave Bioreactor
2.5
95

Viability (%)
2.0
94
1.5

93
1.0

0.5 A 92

0.0 91
0 2 4 6 8 0 2 4 6 8

Culture Time (Days) Culture Time (Days)

3.5 50
T-Flask

C D
Spinner Flask T-Flask
3.0 Spinner Flask
Roller Bottle
Wave Bioreactor 40 Roller Bottle
Wave Bioreactor
2.5
Glucose (g/l)

EPO (mg/l)

2.0 30

1.5
20

1.0

10
0.5

0.0 0
0 2 4 6 8 2 4 7

Culture Time (Days) Culture Time (Days)

Figure 10: Batch cultivation of CHO cells in different cultivation vessels. (A) growth,
(B) viability, (C) glucose consumption and (D) EPO yields. The error bars represent
the standard deviations calculated from the three independent experiments.

Figure 10C shows the glucose consumption, highest consumption was observed in
wave bioreactor (2.8±0.055 g/l, ‘starting medium glucose concentration was 3.2g/l
and the glucose concentration at the end of cultivation was 0.4g/l’), whereas,
1.9±0.048g/l, 2.2±0.06g/l and 1.8±0.045g/l were obtained in spinner, roller and T-
flask. This correlates well with the cell growth and viability profiles, cell growth and
viability were decreased when the glucose level falls below the 1g/l in wave
bioreactor. EPO production also increased in wave bioreactor compared to other
systems (Fig: 10D). The highest EPO production obtained at wave bioreactor was
42±1.16mg/l, this was almost 31.6%, 21.2% and 40% (Student’s t-test p<0.002)
higher than spinner (28.7±0.9mg/l), roller (33.1±0.95mg/l) and static cultures
Improvement of Recombinant CHO Cell Growth, Comparison 1018

(25.2±0.65mg/l). The overall observation with different culture vessel conditions, cell
growth and EPO production were highly increased in wave bioreactor, this may be
due to the continuous aeration and good agitation. Hence, the wave bioreactor was
chosen for the higher yields of cell growth and protein expression. These results
indicate the continuous aeration and good agitation could increase CHO cell growth
and EPO production significantly. Decrease in cell growth and protein production in
T-flask (no agitation and only head space aeration), spinner flask and roller bottles
(continuous agitation, but both systems are oxygenated by gas diffusion through the
head space) may be due to limitation in aeration and agitation. Mammalian cells
require dissolved oxygen for growth and metabolism, but due to the low solubility of
O2 in culture medium, a continuous supply of O2 from the gas phase is required[42].
Cell growth and EPO production was significantly increased in roller bottles than
in spinner flasks. Cultures in spinner flasks frequently show signs of oxygen
limitation, especially at high cell densities[43]; this could be the one reason to
decrease cell growth and EPO yields in spinner flasks, whereas in roller bottles,
medium interact with head space oxygen properly due to the rotary motion of rollers,
which can improve the dissolved oxygen level better than in spinner flask.
Additionally, roller bottles were opened (up to one screw) for the better supply of
atmospheric oxygen.

Conclusions
Gradual reduction of serum is essential to achieve fully adapted serum-free cultivation
of CHO cells. Since all commercially available media will not support equally to all
cell lines growth, it may be essential to select the best commercially available medium
for growth and protein expression. Glutamine replacement with glutamine based
dipeptide (glutamax) decreased ammonia production and enhanced cell growth,
viability and EPO expression. Substitution of glutamine with glutamax is an added
advantage for the commercial production of EPO by rCHO cells. Addition of insulin
to serum-free cultivation of CHO cells improved the cell growth, viability, protein
production and suppresses cell death. Appropriate timely repeat addition of insulin
could suppress cell death more effectively and improve the protein production than
single addition. CHO cell growth and EPO production was significantly increased in
wave bioreactor by continuous supply of aeration and sufficient agitation than in static
condition (T-flask), spinner flask and roller bottle. This work may be useful for
further study on a large scale process for highly efficient production of proteins
toward industrial applications. The information obtained in this work can be applied
to other cell cultures, which have wide utilization and application.
1019 Rambabu Surabattula et al

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