Optimized Feeding Strategy for NS0 Cells
Jennifer Walowitz, Lia Tescione, William Paul, David Jayme and Stephen Gorfien
Invitrogen Corporation, 3175 Staley Road, Grand Island, New York 14072 U.S.A.
% of Theoretical
5.00e+6
250.00
5.00e+6
250.00
4.00e+6
200.00
4.00e+6
200.00
3.00e+6
150.00
3.00e+6
150.00
2.00e+6
100.00
2.00e+6
100.00
1.00e+6
50.00
60.00
2.00e+6
40.00
1.00e+6
Day 4
Day 6
L-Cystine*
59
ND
ND
L-Glutamine
27
ND
ND
L-Leucine
76
25
10
L-Methionine
83
24
10
L-Tyrosine
78
38
25
20.00
0.00
0.00
2
3
4
5
Time (days)
6
7
8
IgG production in Catalog
IgG production in Low G/G
Viable cell density in Catalog
Viable cell density in Low G/G
L-Valine
33
81
0.00
0.00
2
20
4
5
6
7
Time (days)
8
9
50.00
1.00e+6
0.00
0.00
10
2
* The amount of L-cystine cannot be accurately quantified
in the presence of L-cysteine. L-cystine and L-cysteine
have the same retention times and this results in a single
peak.
3
4
5
6
7
8
9
10
Time (days)
IgG production: fed 1 mM GLN, 5AA, 1000X CLC
IgG production: fed 2 mM GLN, 5AA, 1000X CLC
Viable cell density: fed 1 mM GLN, 5AA, 1000X CLC
Viable cell density: fed 2 mM GLN, 5AA, 1000X CLC
IgG production: fed 1 mM GLN, 5AA, 1000X CLC
IgG production: fed 2 mM GLN, 5AA, 1000X CLC
Viable cell density: fed 1 mM GLN, 5AA, 1000X CLC
Viable cell density: fed 2 mM GLN, 5AA, 1000X CLC
Figure 4: rNS0 peak IgG expression levels were similar when glutamine was increased from 1 mM to 2 mM in shake
flask cultures. Peak expression levels were reduced in bioreactor cultures fed with 2 mM glutamine.
Table I: Several amino acids were reduced during the
bioreactor run with low glucose/low glutamine
conditions.
Figure 1: rNS0 cell growth and IgG production were
similar in catalog and low glucose/low
glutamine medium conditions.
3
IgG production (µg/mL)
3.00e+6
Day 2
IgG production (µg/mL)
80.00
Viable cell density (cells/mL)
100.00
IgG production (µg/mL)
Viable cell density (cells/mL)
Bioreactors
Shake Flasks
120.00
4.00e+6
Recombinant NS0 (rNS0) cells expressing IgG were adapted to growth in a proteinfree, chemically defined medium (CD Hybridoma Medium). Liquid Media
Concentrate (LMC) technology was used to segregate nutrient components into
groupings that favor complete solubility and selective component addition to a culture.
Two pre-solubilized feed subgroups were combined immediately prior to culture
addition resulting in a low salt, concentrated supplement containing selected nutrients
(except L-glutamine). The effects of nutrient feeding to standard CD Hybridoma
Medium, and to a modified (reduced glucose and glutamine) version of CD
Hybridoma Medium were studied. Spent medium analysis was employed to identify
rate-limiting nutrients, which were combined to form a simplified feed supplement.
Other variables included addition of lipid-based feed supplements and comparison of
nutrient supplementation in bioreactors versus shake flask cultures. Results indicate
potential for inhibitory effects of non-critical nutrients and different nutrient utilization
profiles in the controlled environment of a bioreactor versus less controlled shake flask
cultures.
Effect of Glutamine Concentration on rNS0 Cell Growth and IgG Production
in Shake Flasks and Bioreactors in Low Glucose/Low Glutamine Medium
Amino Acid Profile of a Bioreactor Culture
in Low Glucose/Low Glutamine Medium
rNS0 Cell Growth and IgG Production in Bioreactors
in Catalog and Low Glucose/Low Glutamine Medium
5.00e+6
Viable cell density (cells/mL)
ABSTRACT
Growth of NS0 cells in a protein-free, chemically defined culture system has been
made possible by the development of cyclodextrin-based lipid additives. Batch
cultures of NS0 cells in this system demonstrated growth and expression levels equal
to or better than levels obtained in serum supplemented media. To improve the total
yield and the efficiency of recombinant product expression in chemically defined
culture, several nutrient supplementation strategies were tested in cultures of NS0
cells.
rNS0 cell stocks were maintained in low glucose/low glutamine CD Hybridoma Medium supplemented with
250X Cholesterol Lipid Concentrate. Each bioreactor was seeded at 3x105 viable cells/mL in a 4 L working
volume of low glucose/low glutamine medium. Three 250 mL shake flask control cultures were seeded at 3x105
viable cells/mL in 100 mL for each bioreactor. Glucose was maintained at 1 g/L in the bioreactors. Beginning
on day 2, glucose was adjusted daily to 1 g/L in the shake flasks. The initial glutamine concentration was 2 mM
and cells were fed 1 mM or 2 mM glutamine daily beginning on day 2. Cells were fed with a 1:1000 dilution of
Cholesterol Lipid Concentrate on days 3 and 5 and with a 1:100 dilution of 5AA Solution on days 4 and 6.
Samples were taken daily from day 2 through day 10 to determine viable densities and IgG concentrations.
rNS0 cell stocks were maintained in catalog or low glucose/low glutamine CD Hybridoma Medium supplemented
with 250X Cholesterol Lipid Concentrate. Each stock was used to seed a bioreactor at 3x105 viable cells/mL in a
4 L working volume of the corresponding media. For the low glucose/low glutamine condition, glucose was
maintained at 1 g/L. The initial glutamine concentration was 2 mM and cells were fed 1 mM glutamine daily
beginning on day 2. Samples were taken daily from day 2 through day 7 to determine viable densities and IgG
concentrations. Samples from the low glucose/low glutamine bioreactor run were analyzed for amino acids by
HPLC. The amino acid profile indicated that cystine, glutamine, leucine, methionine, tyrosine, and valine were
substantially reduced during the run (other data not shown). When the concentration was below the limit of
detection, it was reported as not detected (ND).
INTRODUCTION
NS0 is a non-immunoglobulin secreting, non-light chain synthesizing subclone of
NS-1. NS0 cells lack the glutamine synthetase (GS) enzyme, making them useful for
recombinant expression systems utilizing GS as a selectable marker (1). Cholesterol
auxotrophy of NS0 cells was demonstrated by Keen and Steward (2). They also
showed that NS0 cells can be adapted to cholesterol independence and grown to high
density in a protein-free medium. This protein-free medium would not support growth
of the original NS0 cells unless further supplemented with cholesterol, phosphatidyl
choline and β cyclodextrin. Once adapted to the modified medium, NS0 cells were
reported to reach peak viable cell densities of about 1.2 x 10 6 /mL, although the lipids
precipitated after one week in the medium. The low peak cell density and
precipitation problems encountered by Keen and Steward led us to investigate
alternate means of sterol supplementation. W e have previously described
development of cyclodextrin-based lipid supplements, which when added to a proteinfree hybridoma culture medium made it possible to grow NS0 cells in a completely
protein-free culture system lacking animal derived components (3). Peak cell density
in this small scale system exceeded previously reported results obtained in systems
which contained animal derived components.
200.00
Bioreactors
% of Theoretical
4.00e+6
150.00
3.00e+6
100.00
2.00e+6
Day 2
50.00
1.00e+6
0.00
% of Theoretical
Day 4
Day 2
1 mM
GLN
2 mM
GLN
1 mM
GLN
2 mM
GLN
L-Cystine*
62
86
42
44
L-Glutamine
42
26
28
L-Leucine
83
88
L-Methionine
84
92
L-Tyrosine
81
L-Valine
86
Day 4
1 mM
GLN
2 mM
GLN
1 mM
GLN
2 mM
GLN
L-Cystine*
60
73
38
ND
40
L-Glutamine
24
10
ND
ND
55
34
L-Leucine
71
77
36
15
53
32
L-Methionine
71
78
35
NQ
89
56
37
L-Tyrosine
71
77
42
17
95
62
44
L-Valine
75
83
45
21
0.00
2
3
4
5
Time (days)
6
7
8
IgG production: fed GLN
IgG production: fed GLN, PNS
IgG production: fed GLN, 5AA
Viable cell density: fed GLN
Viable cell density: fed GLN, PNS
Viable cell density: fed GLN, 5AA
Table II: Amino acid profiles were different in shake flasks and bioreactors at both glutamine concentrations.
Several amino acids were depleted more quickly at 2 mM glutamine than at 1 mM glutamine.
Figure 2: IgG production increased slightly when rNS0 cells in low glucose/low glutamine medium were fed amino
acid supplements.
Samples from the low glucose/low glutamine bioreactors and shake flask controls described in Figure 4 were
analyzed for amino acids by HPLC. On day 4, the samples were taken prior to feeding the 5AA Solution. When
the concentration was below the limit of detection, it was reported as not detected (ND). If an amino acid was
detected but present below the limit of quantitation, it was reported as not quantified (NQ).
rNS0 cell stocks were maintained in low glucose/low glutamine CD Hybridoma Medium supplemented with
250X Cholesterol Lipid Concentrate. Each bioreactor was seeded at 3x105 viable cells/mL in a 4 L working
volume of low glucose/low glutamine medium. Glucose was maintained at 1 g/L. The initial glutamine
concentration was 2 mM and cells were fed 1 mM glutamine daily beginning on day 2. The bioreactors were fed
on day 4 and day 6 with a 1:100 dilution of CD Hybridoma Medium PNS or 5AA Solution. Samples were taken
daily from day 2 through day 7 to determine viable densities and IgG concentrations.
* The amount of L-cystine cannot be accurately quantified in the presence of L-cysteine. L-cystine and Lcysteine have the same retention times and this results in a single peak.
Summary of Nutrient Feeding Strategy Effects on IgG Production
in Bioreactors in Low Glucose/Low Glutamine Medium
Effect of Amino Acid and Lipid Supplements on rNS0 Cell Growth and IgG Production
in Bioreactors in Low Glucose/Low Glutamine Medium
180.00
PNS +/- Cholesterol Lipid Concentrate
5AA +/- Cholesterol Lipid Concentrate
5.00e+6
200.00
5.00e+6
MATERIALS AND METHODS
160.00
200.00
100.00
2.00e+6
50.00
1.00e+6
4.00e+6
150.00
3.00e+6
100.00
2.00e+6
IgG production (µg/mL)
3.00e+6
IgG production (µg/mL)
150.00
Viable cell density (cells/mL)
4.00e+6
IgG production (µg/mL)
140.00
Viable cell density (cells/mL)
A proprietary rNS0 cell line that expresses IgG was used in bioreactor experiments.
This rNS0 line does not utilize the Glutamine Synthetase (GS) expression system.
Shake Flasks
IgG production (µg/mL)
Viable cell density (cells/mL)
5.00e+6
Large-scale production of recombinant proteins is often performed using batch culture
systems, which have the advantages of simplicity and reproducibility over more
complex perfusion culture systems. However, productivity may be limited by
nutrient depletion and/or build-up of toxic metabolites in batch culture systems. W e
have recently described development of a model culture system in Chinese Hamster
Ovary (CHO) cells for optimization of recombinant protein expression through
nutrient supplementation (4). The goal of our present work was to take a similar
approach to improve productivity of an rNS0 line. Several approaches to provide ratelimiting nutrients to the cultures have been tested and indicate that feeding amino acids
and lipids together improve productivity in these cells. Differences in performance
between shake flask and bioreactor environments suggest a need to conduct
optimization studies under conditions of controlled pH and dissolved oxygen.
Cells and Base M edia
Effect of Glutamine Concentration on Amino Acid Profile
in Shake Flasks and Bioreactors in Low Glucose/Low Glutamine Medium
Effect of Amino Acid Supplements on rNS0 Cell Growth and IgG Production
in Bioreactors in Low Glucose/Low Glutamine Medium
50.00
1.00e+6
120.00
100.00
80.00
60.00
40.00
Amino Acid and Lipid Supplements
The CD Hybridoma M edium Partial Nutrient Supplement (PNS) was prepared by
mixing equal volumes of CD Hybridoma M edium Partial Nutrient Supplement Acid
Solubles (GIBCO 00-0336DG) and CD Hybridoma M edium Partial Nutrient
Supplement Base Solubles (GIBCO 00-0337DG) within 10 minutes of use. The Acid
Solubles contain all the amino acids in CD Hybridoma M edium except L-glutamine.
The PNS was added at a 1:100 dilution in amino acid feeding strategies.
CD Hybridoma M edium 5 Amino Acid (5AA) Solution of cystine, leucine, methionine,
tyrosine, and valine was prepared to reduce the number of amino acids in the PNS feed.
A 1:100 dilution of 5AA Solution was mixed with a basic solution within 10 minutes of
use to neutralize the pH.
Cholesterol Lipid Concentrate (CLC), 1000X Aqueous Liquid (SKU # 01-0025DG)
was used in lipid feeding strategies. This cyclodextrin-based lipid concentrate was
added to bioreactors at a 1:1000 dilution.
Cell M aintenance
rNS0 cells were grown in agitated suspension culture in shake flasks on a platform
rotating at 125 rpm. The cells were incubated at 37°C in a humidified atmosphere of
8% CO 2 in air. rNS0 stocks were maintained in catalog and low glucose/low glutamine
media. Stocks were passaged using a three or four day schedule. For the three day
passage, cells were seeded at 3x10 5 viable cells/mL in 100 mL in a 250 mL shake flask.
For the four day passage, cells were seeded at 2x10 5 viable cells/mL in 100 mL in a
250 mL shake flask. On day 2 of the four day schedule, cells in low glucose/low
glutamine medium were fed 2 mM glutamine and glucose was adjusted to 2 g/L. Cells
were cultured in 200 mL in 500 mL shake flasks to scale up for the bioreactor runs.
Bioreactor Experiments
Three 7.5 L CelliGen Plus ® (New Brunswick Scientific Co., Inc., NJ) stirred tank
vessels with a 4 L working volume were used for bioreactor experiments. For each
unit, a pitched blade impeller was used at an agitation speed of 60 rpm. Temperature
was maintained at 37°C. The pH was set at 7.30 and controlled with CO 2 . Dissolved
oxygen was maintained at 25% saturation in air. All gasses were sparged. The initial
gas flow rate was 0.1 L/min and increased as necessary to meet the oxygen demand of
the culture.
rNS0 cells were seeded at 3x10 5 viable cells/mL and aseptically transferred to the
bioreactor. Three 250 mL shake flask control cultures were seeded at 3x10 5 viable
cells/mL in 100 mL for each bioreactor run. For low glucose/low glutamine conditions
in bioreactors, glucose was monitored and controlled on-line at 1 g/L using a YSI
analyzer (YSI Inc., Yellow Springs, OH). The initial glutamine concentration was 2
mM and bioreactors were fed the equivalent of 1 mM glutamine daily beginning on day
2. CD Hybridoma M edium PNS, 5AA Solution, and Cholesterol Lipid Concentrate
were used in fed batch runs as indicated in the figure legends. Cell densities were
determined using an electronic particle counter (M odel Z2 TM , Beckman Coulter, Inc.,
Hialeah, FL) and viabilities were estimated using trypan blue exclusion.
Analytical Methods
Glucose concentrations in shake flask cultures were measured off-line using a YSI
analyzer. Immunoglobulin content was quantitated on a protein G Immunodetection
Sensor Cartridge (2.1 mm x 30 mm) from Applied Biosystems, Inc. Amino acids were
quantitated using a pre-column derivatization followed by reverse phase HPLC.
0.00
8
IgG production: fed GLN, PNS
IgG production: fed GLN, PNS, 1000X CLC
Viable cell density: fed GLN, PNS
Viable cell density: fed GLN, PNS, 1000X CLC
0.00
2
3
4
5
Time (days)
6
7
20.00
8
0.00
IgG production: fed GLN, 5AA
IgG production: fed GLN, 5AA, 1000X CLC
Viable cell density: fed GLN, 5AA
Viable cell density: fed GLN, 5AA, 1000X CLC
1
L
1 ow
5A mM G/G
A G fe
, C LN d
LC ,
7
L
m ow
M G
G /G
LN f
, 5 ed
A
A
6
Ca
ta
lo
4
5
Time (days)
nf
ed
3
g
0.00
2
Lo
1 wG
m /
M G
G fe
LN d
0.00
U
Liquid Media Concentrate (LMC) technology was used to prepare catalog and low
glucose/low glutamine CD Hybridoma M edium (5). All components were added
aseptically. CD Hybridoma 50X Acid Solubles (GIBCO PL000686) were added to
sterile distilled, deionized H 2 O at a 1:50 dilution. Next, CD Hybridoma 25X Base
Solubles (GIBCO PL000687) were added at a 1:25 dilution and CD Hybridoma 50X
Salts (GIBCO PL000717) were added at a 1:50 dilution. Glutamine was supplemented
at 2 mM (low glucose/low glutamine) or 8 mM (catalog) using 200 mM L-glutamine
(GIBCO 25030-081). The 250X Cholesterol Lipid Concentrate (GIBCO 12531-018)
was added at a 1:250 dilution. For bioreactor work, Penicillin-Streptomycin (GIBCO
15140-122) was added at a 1:100 dilution. Glucose levels were adjusted using a 30%
glucose solution.
Feeding strategy
Figure 3: IgG production increased when rNS0 cells in bioreactors were fed amino acid and lipid supplements.
Figure 5: Adapting rNS0 cells from catalog to low glucose/low glutamine medium and feeding amino acid and lipid
supplements resulted in a doubling of IgG levels from the catalog control.
rNS0 cell stocks were maintained in low glucose/low glutamine CD Hybridoma Medium supplemented with
250X Cholesterol Lipid Concentrate. Each bioreactor was seeded at 3x105 viable cells/mL in a 4 L working
volume of low glucose/low glutamine medium. Glucose was maintained at 1 g/L. The initial glutamine
concentration was 2 mM and cells were fed 1 mM glutamine daily beginning on day 2. The bioreactors were fed
with a 1:1000 dilution of Cholesterol Lipid Concentrate on days 3 and 5. On days 4 and 6, the bioreactors were
fed a 1:100 dilution of either CD Hybridoma Medium PNS or 5AA Solution. Samples were taken daily from day
2 through day 7 to determine viable densities and IgG concentrations.
Day 7 IgG concentrations from bioreactor runs were compared to demonstrate the improvement in production
achieved by developing a feeding strategy for rNS0 cells in low glucose/low glutamine medium. In a batch run
with catalog medium, the day 7 IgG production was 82 µg/mL. All other bioreactors were fed-batch runs with
low glucose/low glutamine medium with various supplements. Feeding all three supplements (glutamine, 5AA
Solution, and Cholesterol Lipid Concentrate) resulted in peak IgG production of 164 µg/mL on day 7.
DISCUSSION
W e recently developed a nutrient supplementation strategy to optimize recombinant
protein expression in CHO cells (4). A similar approach was used to improve antibody
production in an rNS0 cell line. However, initial experiments indicated that glucose
and glutamine requirements were vastly different for the CHO and rNS0 systems.
Since this rNS0 cell line lacks the glutamine synthetase (GS) enzyme, these cells
require glutamine supplementation. rNS0 growth and IgG production were similar in
catalog and low glucose/low glutamine media. Less overall ammonia was produced
with the low glucose/low glutamine medium (data not shown), so this condition was
used in all further development work.
Since amino acid supplementation has been used to improve antibody production in
mammalian cell lines (4, 6, 7), CD Hybridoma M edium PNS was used in initial feeding
experiments. A slight improvement in rNS0 production was seen at the end of the run.
In order to reduce the amino acids in the feed, we looked at the amino acid
consumption profile, which indicated that cystine, leucine, methionine, tyrosine, and
valine were substantially reduced during the bioreactor run with low glucose/low
glutamine conditions. A 5AA Solution was prepared with these amino acids. Similar
results were seen with CD Hybridoma M edium PNS or the 5AA Solution, which
suggested that one or more other components might be limiting.
Since the rNS0 line is cholesterol dependent, cells were fed with Cholesterol Lipid
Concentrate in addition to the amino acid supplements. Previous studies indicated that
250X Cholesterol Lipid Concentrate could not be used in feeding strategies because
cyclodextrin levels became toxic to the cells. However, the 1000X Cholesterol Lipid
Concentrate could be used because this supplement contributed less cyclodextrin to the
cultures. The combined amino acid and lipid feed improved IgG production.
Preliminary experiments suggested depletion of fatty acids, yet feeding fatty acids in
addition to 5AA Solution did not improve productivity (data not shown) while feeding
with cholesterol and fatty acids in addition to 5AA Solution improved IgG levels.
Adapting rNS0 cells from catalog to low glucose/low glutamine medium and feeding
with amino acid and lipid supplements resulted in a doubling of IgG levels from the
catalog control. Yet, amino acid analysis revealed that glutamine levels were not
detectable during most of the run, despite daily feeds beginning on day 2. The
glutamine feed was increased to 2 mM, which resulted in faster growth, but lower IgG
levels. Amino acid analysis showed more rapid depletion of the other fed amino acids
in the 2 mM culture. W e hypothesize that the lower amino acid availability resulted in
lower IgG production in the 2 mM bioreactor culture. IgG production was similar at
both glutamine concentrations in shake flask cultures, presumably as a result of less
rapid depletion of amino acids. This suggests that optimization of feeding times and/or
concentrations of other nutrients may further improve IgG production in bioreactors.
IgG production was increased through the development of amino acid and lipid feeding
strategies in low glucose/low glutamine medium. This work has established the basis
for further optimization studies in this system. While off-line HPLC analysis and a
bolus feeding strategy were used in this work, on-line analysis can be used to
continuously monitor depletion of amino acids and optimize feeding strategies (8). W e
are working to develop an on-line monitoring system to optimize nutrient
concentrations and feeding rates.
SUMMARY
•
rNS0 cell growth and IgG production in low glucose/low glutamine medium was
comparable to catalog control medium.
•
IgG production was improved by developing a nutrient feeding strategy for rNS0
cells in low glucose/low glutamine medium.
•
•
A slight increase in IgG production was seen when amino acid supplements
were fed.
•
IgG levels were doubled when a combined feed of amino acid and lipid
supplements was used.
Optimization of nutrient concentrations and feeding times may further improve
rNS0 IgG production.
ACKNOW LEDGEMENTS
The authors wish to thank the members of the Invitrogen M edia Analytical Laboratory
for their assistance in IgG and nutrient quantitation. W e also thank Delia Fernandez for
for her assistance in bioreactor preparation.
CelliGen Plus ® is a registered trademark of New Brunswick Scientific Co., Inc.
Z2 TM is a trademark of Beckman Coulter, Inc.
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