Process Development
Optimized Nutrient Additives
for Fed-Batch Cultures
utrient media designed for highdensity animal cell cultures have
historically contained relatively
high levels of glucose and
glutamine, because those media
constituents were presumed to be the
primary metabolic energy sources. Yet
rapid catabolism of these nutrients,
particularly under conditions of inadequate
oxygenation, leads to accumulation of
lactate and ammonia — and these
metabolic wastes can limit cell growth and
productivity.
Large-scale production of recombinant
proteins often uses batch culture systems.
Batch cultures are simpler and more
reproducible than the more complex
perfusion-culture systems. Nutrient
supplements are frequently added to cell
cultures used for biopharmaceutical
production to extend culture life and
improve yield. The productivity of fed-batch
systems, however, can be limited by nutrient
depletion or by a build-up of toxic
metabolites or both.
We developed a model fed-batch system
for mammalian cell culture. By adapting
cell lines to reduced glucose and glutamine
medium and by optimizing the nutrient
supplement, we were able to boost nutrient
availability as the cells transitioned from
growth to expression. Our model exhibited
sustained maximal cell density and
enhanced expression by reducing
metabolic waste products, minimizing
osmolality increases, and providing critical
nutrients.
N
Stephen F. Gorfien,
William Paul, David Judd,
Lia Tescione, and
David W. Jayme
Cell density, longevity, and
expression are the economics of
cell cultures in biopharmaceutical
production — and rich growth
media are the investment.
Glucose and glutamine are
primary energy sources for culture
growth, but their associated
metabolic wastes can actually
harm cell cultures. Adapting cell
lines to reduced levels of
glucose and glutamine, then
feeding the cultures chemically
defined nutrient supplements,
increases cell viability,
maximizes cell density, and
increases product expression.
Too Rich Can Be a Problem
Corresponding author Stephen F. Gorfien is
director of industrial applications; and William Paul
is senior research associate, David Judd and
Lia Tescione are scientists, and David W. Jayme is
senior director in the cell culture R&D department at
GIBCO Cell Culture R&D, Invitrogen Corporation,
3175 Staley Road, Grand Island, NY 14072,
800.828.6686 ext. 46633, fax 716.774.6996,
[email protected], www.invitrogen.com.
34
BioPharm International
APRIL 2003
Many serum-free and protein-free culture
media are available commercially, and
most are rich in nutrients — particularly in
glucose and glutamine. However, as these
nutrients feed the cells, the byproducts
from the energy transfer can be detrimental
to cell growth, and this may be particularly
true of the byproducts of glucose and
glutamine metabolism (1,2). Attempts at
supplementing an already rich batchculture medium with additional glucose
and glutamine can exacerbate the problem
of potentially toxic metabolite
accumulation (3). Figure 1 shows that
media with reduced levels of glucose and
glutamine prolonged viability by at least
two days (Figure 1a) and improved
r-galactosidase (rGal) expression in a
recombinant Chinese hamster ovary
(rCHO) cell line (Figure 1b).
The problem with glucose. High levels of
glucose can result in high levels of lactate
through glycolysis. Lactate accumulation
can reduce the pH throughout the culture,
and that low pH can be detrimental to cell
viability and productivity (1). This
problem can be addressed by adding a base
to the culture in a bioreactor. Within a
pH-controlled bioreactor, neutralizing
metabolic acids can increase osmolality.
Although elevated osmolality has been
shown to improve productivity in some
mammalian systems (4), an osmolality
increase that is too rapid and too high can
negatively affect growth and protein
expression, limiting bioreactor longevity in
fed-batch mode. Figure 2a shows that far
less lactate is produced when there is less
glucose in the medium. Lactate in the low
glucose and glutamine culture still
increases because these cultures are fed
with glucose to maintain the low level.
The problem with glutamine. High levels of
glutamine in culture media can cause
ammonia to accumulate. The ammonia
results from either metabolic hydrolysis to
glutamic acid or from spontaneous
deamidation as a result of medium storage.
Reduced expression of recombinant tissue
plasminogen activator (rtPA) has been
associated with elevated ammonia
concentration (5), and ammonium chloride
was shown to inhibit multiplication of
herpes simplex virus type 1 in Vero cells
(6). Ammonia has also been shown to
(a)
(a)
Catalog
1.20
Catalog
Low glucose/Low glutamine
2.0
1.5
1.0
0.5
0.0
3
4
5
6
7
8
9
10
11
0.80
0.60
0.40
0.20
0.00
12
3
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Catalog
(b)
Low glucose/Low glutamine
0.16
Ammonia (mg/106 Cells)
rgal (U/mL)
(b)
Low glucose/Low glutamine
1.00
2.5
Lactate (mg/106 Cells)
Viable Cell Density (106)
3.0
4
5
6
7
8
9
10
11
4
Catalog
5
6
7
8
7
8
Low glucose/Low glutamine
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
12
3
Day
4
5
6
Day
Figure 1. Comparison of rCHO cultures adapted for growth in a
Figure 2. Improved cell density and productivity of rCHO cultures
reduced glucose and glutamine and a high glucose and glutamine
(catalog control) media in (a) viable cell density and (b) rGal
expression. Results show prolonged cell viability (days 10–12)
and improved production throughout with the reduced glucose
and glutamine mixture.
adapted to a reduced glucose and glutamine medium show an
associated, significant reduction in (a) lactate from glycolysis and
(b) ammonia from metabolic hydrolysis to glutamic acid.
affect glycosylation of a recombinant
protein (7). Figure 2b shows the much
smaller ammonia production from a
medium that is low in glutamine.
The benefit of partial nutrient supplements. To
minimize these toxicity problems while
increasing productivity, we simplified a
chemically defined (CD), protein-free
medium for CHO cells (CD CHO Medium)
by reducing the levels of glucose and
glutamine. We tested several approaches to
providing rate-limiting nutrients to the
cultures. We developed a concentrated, lowsalt, partial nutrient supplement (PNS) to
improve nutrient availability to rCHO cells.
Figure 3 shows, however, that feeding cells
cultured in reduced glucose and glutamine
CD CHO Medium with PNS on day 3
postplanting did not improve productivity or
viability. (See the “Materials and Methods”
sidebar for a complete description of the
processes).
The benefit of an optimized feed. By
analyzing spent media, we identified the
amino acids (asparagine or aspartic acid,
glutamine or glutamic acid, and cystine)
depleted during culture (Table 1). To
minimize ammonia generation, we fed
glutamic acid and aspartic acid to the
cultures rather than glutamine and
asparagine and added cystine as well. The
addition of these glycogenic amino acids
corrected the rate-limiting deficiencies
(Table 1). We also analyzed shake flasks
versus bioreactors, and whether more
frequent feeding of the amino acid
supplements (but at a lower concentration
per feed) improved productivity (see the
“Materials and Methods” sidebar).
Feed Only What Is Needed
We found that reducing glucose and
glutamine extended rCHO cell viability
and production levels in a fed-batch
culture system and that adding a simple,
optimized nutrient feed consisting of only
three amino acids further enhanced total
production.
Zhou and coworkers have reported a
metabolic shift in hybridoma cells adapted
to grow in medium containing reduced
levels of glucose and glutamine (8). Our
studies — using rCHO cells engineered to
overexpress rGal (9) (Figure 4) or human
immunoglobulin G (rIgG) (Figure 5) and
adapted to reduced glucose and glutamine
conditions — confirm those observations
and illustrate the benefits of fed-batch
supplementation of a defined cocktail of
nutrient additives to extend bioreactor
longevity and enhance product yield.
A metabolic shift model. CHO cells adapted
to reduced glucose and glutamine
conditions exhibit a reduced metabolic
requirement for these nutrients. Reduced
consumption is accompanied by a reduced
accumulation of lactate and ammonia. Our
study shows an increase in the production
of rGal or rIgG under these growth
conditions, which suggests a more efficient
conversion of nutrient to product. This is
in contrast to results described by
Altamirano et al. (10) using a continuous
culture of rCHO cells expressing rtPA.
They found that rtPA production decreased
as glucose supply to the culture was
BioPharm International
APRIL 2003
35
Process Development
Materials and Methods
Our goal was to determine if reduced glucose
and glutamine extended recombinant Chinese
hamster ovary (rCHO) growth and production
in a fed-batch culture system. The materials
and methods we used are described here;
results are discussed in the text.
Cells and media. Recombinant CHO cells
engineered to overexpress -galactosidase
(9) or human rIgG (proprietary cell line) were
rapidly adapted to growth in either CD CHO
Medium (GIBCO, Invitrogen Corporation)
supplemented with 8.0 mM glutamine or a
modified CD CHO Medium prepared with
2.0 g/L glucose and supplemented with
glutamine in varying concentrations between
2.0 and 8.0 mM.
Nutrient supplements. A CD CHO
Medium–based partial nutrient supplement
(PNS) containing all the amino acids of the
standard CD CHO Medium formulation,
except glutamine, was prepared as a 100-fold
acid-soluble concentrate. A companion basesoluble solution of sufficient concentration to
neutralize the acid group was also prepared.
Partial amino acid supplements consisting of
amino acids determined to be necessary for
continued growth or for protein expression
were prepared as approximately
4,000–6,000-fold concentrates. A 30%
glucose solution was prepared in distilled
water (dH2O). A stock solution of
0.1 g NaCl/mL dH2O was used to adjust
culture osmolality and volume in some control
cultures non-PNS fed.
Analytical assays. A precolumn derivatization
(Accutag, Waters Corporation) followed by
reverse-phase HPLC was used to quantitate
amino acids and ammonia in culture media
samples using a 474 scanning fluorescence
detector (Waters). Glucose and lactate were
measured by a YSI analyzer (YSI Inc.).
reduced, and they postulated that the high
growth rate imposed by the culture dilution
rate in a continuous system forced the cells
to derive energy sources at the expense of
dispensable metabolites (such as rtPA).
In our low glucose and glutamine system,
cell growth rate in shake flasks was not
increased over the high glucose and
glutamine system, which supports a
metabolic shift model. It is not clear from
these results whether the reduced glucose
36
BioPharm International
APRIL 2003
Recombinant -galactosidase (rGal)
activity was measured in cell lysates using a
modification of the methods described by
Hall et al. (13) and Miller (14). Briefly,
1.0 mL of cell suspension was collected
from each sample, subjected to four
freeze–thaw cycles, and centrifuged.
A rGal standard (0.0008 mg/mL) was
added to the first two rows of a 96-well plate
on ice to generate a standard curve of
0–8 ng/mL rGal (0–10 L of standard per
well). Cell extract (10 L per well) was
added to each of the remaining wells. Each
well received 100 L of a cocktail consisting
of 77 L of 0.1 M of sodium phosphate
(pH 7.5) and 22 L of 4 mg/mL
o-nitrophenyl--D-galactopyranoside
(ONPG) at pH 7.5 and containing 2 mM of
-mercaptoethanol and 1 L of 100-fold
solution of 0.1 M MgCl2 and 4.5 M of
-mercaptoethanol.
A 26-minute incubation at 37°C was
necessary for a bright yellow color to
develop, and 150 L of 1 M sodium
carbonate was added to each well to stop
the reaction. A microplate reader measured
absorbance at 405 nm (A405).
rIgG analysis was performed using SDS-PAGE
and image analysis. Samples were prepared
according to manufacturers instructions
(Invitrogen, gels and stains are also from this
company) and separated on a 4–12%
Bis–Tris acrylamide gradient gel at 200 V for
35 minutes. Gels were then washed three
times in dH2O for five minutes and stained in
SimplyBlue SafeStain for one hour. The gel
was then washed for one hour in dH2O and
then in a second wash of dH2O for
3–24 hours. Image analysis was performed
using a MDS290 digital camera with a onedimensional (1-D) image analysis software
and glutamine directly precipitate the
metabolic shift or the effect is a result of
reduced production of cytotoxic catabolites
(principally ammonia).
In bioreactors, cells adapted to reduced
glucose and glutamine reached a higher
peak viable cell density than cells adapted
to high glucose and glutamine (Figure 4).
This still suggests a metabolic shift model
because the peak production levels did not
package (both from Kodak Scientific Imaging
Systems). Band densities were compared to
band densities of a known standard.
Cell cultures. rCHO cells were grown in an
agitated suspension culture in 125-, 250-, or
500-mL shake flasks on orbital shaker
platforms rotating at 120–130 rpm. Culture
volumes varied from 20–200 mL, depending
on the flask size. Cultures were incubated at
37°C in humidified atmosphere of 8% CO2
in air. Cells were seeded at a density of
2.0–3.0 105/mL and subcultured at
intervals of three to four days. For low
glucose and glutamine shake flask
experiments, glucose was added daily to
1.0 or 1.5 g/L starting on day 3; no
additional glutamine was supplemented.
Various amino acid supplements were
initially added on day 3 or day 4 when the
viable cell density had reached at least
1 106/mL.
In some experiments, a sterile saline
solution was added to control cultures to
adjust osmolality and volume so that it was
similar to that of PNS-fed cultures. Cell
densities were determined using an
electronic particle counter (Coulter
Electronics). Cell viabilities were estimated
by trypan blue exclusion. Results are
expressed as mean standard deviation
() for replicate cultures.
For some experiments, cells were cultured
in a 7.5-L (4-L working volume) CelliGen
Plus bioreactor (New Brunswick Scientific).
Initial bioreactor control parameters were set
at pH 7.3, dissolved oxygen at 50% air
saturation, and impeller speed at 60 rpm.
Glucose was maintained in low glucose, low
glutamine bioreactors at 0.1 g/L by
automatic addition of glucose concentrate
when triggered by YSI measurement.
occur until after the cells had reached peak
density. Future experiments will include
ammonia measurement in bioreactor
cultures.
Defining the right nutrient cocktails. Many fedbatch additives have been reported to boost
cell density or production (11). These
nutrient cocktails often contain a complete
basal formulation without the principal
inorganic salt constituents (12). Our data
suggest that for some cell systems, such a
Table 1. Spent media was analyzed to identify the amino acids depleted during culture.
Cells adapted to reduced glucose and
glutamine and grown in a stirred tank
reactor showed improved growth and
production compared to shake flask
cultures.
complex nutrient feed may be suboptimal
and may even inhibit both cell
proliferation and protein production.
Analysis of spent culture fluids (Table 1)
illustrates that a simple cocktail of three
amino acids (glutamate, aspartate, and
cystine) sustains cell viability and
productivity under constant low glucose
conditions without further addition of
glutamine (minimal residual levels of
glutamine remain after a seven-day
incubation at elevated cell density).
Following transamination to tricarboxylic
acid (TCA) cycle intermediates, the
carbohydrate backbones of aspartate and
glutamate may be efficiently aerobically
metabolized. Studies are in progress to
investigate coordinate induction of
glutamine synthetase, glutamate
dehydrogenase, or other relevant enzyme
systems under these culture conditions.
In the cell system we used, adding
aspartate and glutamate was necessary but
insufficient for optimal protein yield.
Spent medium analysis indicated that
exogenous cystine was also required. The
nutrient additive containing all three amino
acids yielded significantly higher
Amino Acid Profilea
PNS
A/G
A/G/C
62
153
79
82
8
96
6
7
acid
49
162
121
124
2HCl
0
81
0
79
8
14
7
7
L-arginine
L-aspartic
L-cystine
Control
HCl
L-asparagine
H2O
L-glutamine
L-glutamic
acid
58
158
148
150
L-histidine
HCl H2O
64
162
99
100
Hydroxy-L-proline
74
177
142
147
L-isoleucine
65
159
69
72
L-leucine
62
152
64
67
L-lysine
79
169
55
54
HCl
L-methionine
61
160
71
73
1,372
1,599
1,305
1.323
L-phenylalanine
57
168
90
93
L-proline
72
161
108
109
L-serine
46
134
39
40
Ammonia
a
Amino acids are presented as a percentage of the base media.
(a)
Viable Cell Density (106 Cells)
Peak cell density and rGal expression
were improved when the reduced glucose
and glutamine cultures were fed a
supplement of three amino acids
(glutamate, aspartate, and cystine).
Feeding the three amino acids more
frequently (but at a lower concentration per
feed) resulted in a slightly greater
production improvement than a less
frequent, higher concentration per feed
strategy.
These data present the amino acid profile on day 7 for reduced glucose and glutamine
cultures that were unsupplemented (control), supplemented with partial nutrient
supplement (PNS), supplemented with aspartic and glutamic acid (A/G), and
supplemented with aspartate, glutamate, and cystine (A/G/C).
(b)
rGal (U/mL)
Results are discussed more fully in the text.
To summarize, reducing the concentration
of glucose and glutamine in CD CHO
Medium increased cell viability and
improved production in an rCHO cell line.
The improved performance was
associated with a reduction of lactate and
ammonia metabolic wastes.
Catalog PNS d3
Low glucose/low
glutamine PNS d3
Catalog control
Low glucose/low
glutamine control
3.0
2.5
2.0
1.5
1.0
0.5
0.0
3
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
4
5
6
Catalog control
Low glucose/low
glutamine control
4
5
6
7
8
9
10
11
12
Catalog PNS d3
Low glucose/low
glutamine PNS d3
7
8
Day
9
10
11
12
Figure 3. Feeding a partial nutrient supplement (containing all amino acids, except glutamine,
in a standard media formulation) to cells adapted to low glucose and glutamine did not
significantly improve (a) cell density or (b) product expression when compared with cells
similarly adapted without the supplement suggesting that the supplement may contain
unnecessary or inhibitory components that adversely affect growth and production.
BioPharm International
APRIL 2003
37
Process Development
rGal (U/mL)
(b)
3.0
Catalog bioreactor 3 amino acids
2.5
Low glucose/Low
glutamine bioreactor
3 amino acids
(a)
1.5
1.0
0.5
0.0
3
4
5
6
7
8
9
2.5
Catalog bioreactor 3 amino acids
2.0
Low glucose/Low
glutamine bioreactor
3 amino acids
10
11
12
(b)
1.5
1.0
0.5
10
Viable Cell Density (106 )
2.0
Catalog shake flask
Low glucose/Low glutamine bioreactor IgG
Low glucose/Low glutamine shake flask
8
6
4
2
0
Recombinant Immunoglobolin
(g/mL)
Viable Cell Density (106 )
(a)
160
140
120
100
80
60
40
20
0
2
3
4
5
6
7
8
9
10
11
2
3
reduced glucose and glutamine medium improved in (a) cell
density and longevity, and in (b) rGal expression when fed a
nutrient supplement of three amino acids in comparison to a
catalog (high glucose and glutamine) medium fed with the same
three amino acids.
Bioreactors versus shake flask cultures.
Improved peak cell density and production
in a stirred-tank bioreactor system
compared to shake flask cultures illustrate
the importance of pH and dissolved
oxygen control in nutrient use (Figure 5).
Shifting cells to more efficient aerobic
metabolism through adaptation to reduced
glucose and glutamine conditions would be
expected to increase the requirement for
aspartate and glutamate as precursors to
38
BioPharm International
APRIL 2003
7
8
9
10
11
4
5
6
7
8
9
10
11
Figure 5. Cells grown in a reduced glucose and glutamine
Figure 4. Recombinant CHO cultures grown in bioreactors in
performance than cocktails of the full basal
constituent mixture or of aspartate and
glutamate alone.
Delivery frequency also contributes to an
optimal biological performance. With total
molar amounts of aspartate and glutamate
kept constant, protein production was
improved when the supplement was
delivered more frequently in smaller doses
rather than in two larger additions
(Figure 6a). We further demonstrated (using
a saline control) that the enhanced
productivity from the simple amino acid
cocktail was not simply a consequence of an
osmotic boost (Figure 6b).
6
Day
12
Day
5
Catalog shake flask
Low glucose/Low glutamine bioreactor IgG
Low glucose/Low glutamine shake flask
0.0
3
4
chemically defined CHO medium and fed a nutrient supplement of
only three amino acids show (a) improved growth and (b)
increased rIgG production when grown in shake flasks and even
greater improvement in (a) density and (b) expression when
grown in a bioreactor in comparison to cells grown in shake flasks
in a chemically defined CHO medium (catalog) with high glucose
and glutamine fed with the same three amino acids.
TCA cycle intermediates. In the presence
of adequate levels of aspartate and
glutamate, oxygen availability would then
become the rate-limiting factor. We plan
further studies examining oxygen and
amino acid consumption rates to address
this issue.
Simpler and More Productive
Our data suggest a qualitative difference in the
design of nutrient media intended for fedbatch (and perhaps perfusion) culture
applications and reinforce the potential benefit
of adapting production cells, master cell
banks, and master working cell banks to low
glucose and glutamine culture conditions.
Using an iterative approach, we
developed a model fed-batch system for
mammalian cell culture. Recombinant CHO
cell lines that express rGal or human rIgG
were adapted to a chemically defined,
protein-free medium (CD CHO Medium). A
low salt, concentrated supplement
containing selected nutrients was added to
boost nutrient availability as the cells
transitioned from growth to expression.
Two presolubilized feed subgroups were
combined immediately before being added
to the culture. Nutrient use was determined
by analysis of spent media samples to
simplify the feed supplement. Cells
adapted to reduced levels of glucose and
glutamine exhibited sustained maximal cell
density and enhanced expression. We
added a simplified supplement (glutamate,
aspartate, and cystine) at various times to
determine the optimal conditions for
protein expression.
The reduced glucose and glutamine CD
CHO Medium reduced metabolic waste
products (ammonia and lactate) and
minimized osmolality increases. The
ability of rCHO cells to grow and express
well in a low glutamine basal medium fed
only with glutamate, aspartate, and cystine
suggests that alternate metabolic pathways
can be exploited to improve culture
performance. Additional efficiencies may
be possible by further simplifying the basal
medium or modifying the feeding
schedule. BPI
Process Development
Gal - A/G/C (.5x) d3,6
vc/mL - Control
vc/mL - A/G (.25x) d3,4,6,8/C
(1x) d3,6
Gal - control
Gal - A/G (.25x)d3,4,6,8/C(1x) d3,6
vc/mL - A/G/C (.5x) d3,6
(a)
0.6
2.5
0.5
2.0
Acknowledgments
The authors wish to thank the members of the
Invitrogen Media Analytical Services Laboratory for
their assistance in nutrient quantitation.
References
(1) Hassell, T., Gleave, S., and Butler, M.,
“Growth Inhibition in Animal Cell Culture:
The Effect of Lactate and Ammonia,” Appl.
Biochem. Biotechnol. 30, 29–41 (1991).
(2) Heeneman, S., Deutz, N.E.P., and Buurman,
W.A. “The Concentrations of Glutamine and
Ammonia in Commercially Available Cell
Culture Media,” J. Immunol. Methods 166,
85–91 (1993).
(3) Fike, R. et al., “Feeding Strategies for
Enhanced Hybridoma Productivity: Automated
Concentrate Supplementation,” BioPharm
6(8), 49–54 (1993).
(4) Chua, F.K., Yap, M.G., and Oh, S.K.,
“Hyperstimulation of Monoclonal Antibody
Production by High Osmolarity Stress in eRDF
Medium,” J. Biotechnol. 37(3), 265–275
(15 November 1994).
40
BioPharm International
APRIL 2003
1.0
0.2
0.5
0.1
0.0
3
4
5
6
7
8
9
10
11
0.0
12
Day
Gal (Saline d3,6)
vc/mL (Saline d3,6)
2.5
Gal (PNS d3,6)
vc/mL (PNS d3,6)
0.20
2.0
0.15
1.5
0.10
1.0
0.05
0.5
0.00
3
4
5
6
7
8
9
10
11
12
13
vc/mL 106
(b) 0.25
Gal U/mL
Figure 6. (a) We have done some
work on the effect of more frequent, but
quantitatively less, nutrient feeding:
These preliminary data show that cultures
fed a supplement of three amino acids
(aspartate, glutamate, and cystine) at onequarter strength on four days (with cystine
only added twice) performed better than
the two-day feeding of the three amino
acids, and both showed better density
(lines) and expression (bars) than the
control (unsupplemented); and (b)
to confirm that the boost in expression
(bars) and cell density (lines) was a result
of the nutrient supplement and not a
result of the increased osmolality (salt
concentration), we used cells adapted to
reduced glucose and glutamine and
compared cultures fed saline with those
fed a partial nutrient supplement: These
data show that the improved culture
performance was not a result of
increased osmolality.
1.5
0.3
vc/mL 106
Gal U/mL
0.4
0.0
Day
(5) Hansen, H.A. and Emborg, C., “Influence of
Ammonium on Growth, Metabolism, and
Productivity of a Continuous Suspension
Chinese Hamster Ovary Cell Culture,”
Biotechnol. Prog. 10, 121–124 (1994).
(6) Koyama, A.H. and Uchida, T., “The Effect of
Ammonium Chloride on the Multiplication of
Herpes Simplex Virus Type 1 in Vero Cells,”
Virus Res. 13(4), 271–281 (1988).
(7) Yang, M. and Butler, M., “Effect of Ammonia
on the Glycosylation of Human Recombinant
Erythropoietin in Culture,” Biotechnol. Prog.
16, 751–759 (2000).
(8) Zhou, W. et al., “Alteration of Mammalian
Cell Metabolism by Dynamic Nutrient
Feeding,” Cytotechnol. 24, 99–108 (1997).
(9) Gorfien, S.F. et al., “Recombinant Protein
Production by CHO Cells Cultured in a
Chemically Defined Medium,” Animal Cell
Technology: Basic and Applied Aspects,
Vol. 9, K. Nagai and M. Wachi Eds. (Kluwer
Academic Publishers, New York, 1998),
pp. 247–252.
(10) Altamirano, C. et al., “Analysis of CHO Cells
Metabolic Redistribution in a Glutamate-Based
(11)
(12)
(13)
(14)
Defined Medium in Continuous Culture,”
Biotechnol. Prog. 17, 1032–1041 (2001).
Bibila, T.A. and Robinson, D.K., “In Pursuit of
the Optimal Fed-Batch Process for
Monoclonal Antibody Production,”
Biotechnol. Prog. 11(1), 1–13
(January–February 1995).
Sauer, P.W. et al., “A High-Yielding, Generic
Fed-Batch Cell Culture Process for Production
of Recombinant Antibodies,” Biotechnol.
Bioeng. 67(5), 585–597 (2000).
Hall, C.V. et al., “Expression and Regulation
of Escherichia Coli LacZ Gene Fusions in
Mammalian Cells,” J. Mol. Appl. Genetics 2,
101–109 (1983).
Miller, J.H., “Experiments in Molecular
Genetics,” (Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY, 1972).