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Application-Specific Technical Information—Application Notes
Chinese Hamster Ovary Cells for the Production
of Recombinant Glycoproteins
Camire, Joseph. Art to Science, Vol. 19, No. 1; Logan, UT, 2000
Chinese Hamster Ovary (CHO) cells are a
valuable tool for producing recombinant proteins
with "native" mammalian glycosylation patterns.
Following their introduction in the 1950s, CHO
cell lines have become a workhorse for
manufacturing recombinant proteins. Genetic
engineers can modify CHO cells to produce
proteins with "mammalian" post-translational
modifications, and through gene amplification
can selectively produce high levels of
recombinant proteins.
Process development, using CHO cell lines,
focuses on achieving the maximum amount of
active product. Optimization of the amount of
active product can be achieved in at least two
basic ways. The first way is to increase the
specific productivity (i.e., the product per cell)
through cell line development. Cell line
development may include both sub-cloning the
cell line to select higher producing clones and
use of gene amplification. Drug resistance has
been the tool of choice in the biopharmaceutical
industry to induce gene amplification.
By combining the gene of interest with a
selectable gene, increased production levels
can be accomplished. Using productivity-per-cell
approaches, scientists have increased
expression levels more than 1,000-fold
(Schimke, 1982). Two gene amplification
systems used in CHO cells are the dihydrofolate
reductase (DHFR) system using methotrexate
(MTX) resistance, and the glutamine synthetase
(GS) system using methionine sulfoxamine
(MSX) resistance. The DHFR enzyme catalyzes
the conversion of folate to tetrahydrofolate
(Figure 1).
This precursor is necessary for the de novo
synthesis of purines, pyrimidines, and glycine
(Goeddel, 1990). Methotrexate is a drug which
is similar (i.e., an analog) to folate. MTX binds
to DHFR, thereby inhibiting the production of
tetrahydrofolate. With insufficient levels of
DHFR, cells are deprived of nucleoside
precursors (hypoxanthine and thymidine) and
die. Gene amplification is a technique used by
molecular biologists. They transform cells with
recombinant DNA consisting of the gene of
interest closely linked to the gene for DHFR.
Then during gene amplification the cells are
cultured in increasingly higher levels of MTX.
Those CHO cells that have increased copies of
the DHFR gene, and therefore higher levels of
the enzyme, are selected.
The GS/MSX system is similar to the
DHFR/MTX system. The GS enzyme catalyzes
the production of glutamine from glutamate and
ammonia (Figure 2). Methionine sulfoxamine
binds to the GS enzyme and prevents the
production of glutamine. Gene amplification
occurs when cells are subjected to increasing
concentrations of MSX.
A second way to achieve higher recombinant
protein yields is to increase the cell yield (i.e.,
cells per volume) of the process. This may be
accomplished through process development
(e.g., batch, fed-batch, perfusion, etc.) and
medium development. By increasing the cells
per volume per day, higher levels of product
may be produced.
To demonstrate the utility of this protein
production system, we used CHO cell line
(ATCC® CRL-10154 CHO 5/9 M! 3-18), which
produces human macrophage colony stimulating
factor (hm-CSF). This cell line was produced
using a DHFR-CHO DG44 cell clone as the
parental line. Gene amplification was
performed using the DHFR/MTX system.
The cell growth and protein production kinetics
of this cell line were studied using a powdered
medium developed for CHO cells by
Themo Scientific HyClone. Standard
culturing procedures for CHO cells
were used for these evaluations
(Table 1).
Initial evaluations compared
commercially available serum-free
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and protein-free media to Themo Scientific
HyClone PF-CHO MPSq (SH30333) using the
CHO 5/9 M! 3-18 cell line. Representative
results obtained in these comparisons are
shown in Table 2 and Figure 3.
Table 1: Evaluation Parameters for
use with Shaker and Spinner Cultures
Temperature
CO2 Atmosphere
Agitation
Oxygen
Buffering
37°C
5%
Shaker 100–130 rpm
Spinner 60–80 rpm
Atmospheric
2 g/L Sodium
Bicarbonate
Table 2: Comparison of SH30333 vs
Competitor Medium
Parameter
Maximum Cell
Density (Mean)
Maximum
Population Doubling
Time (Mean)
Maximum Protein
Production (Mean)
SH30333
212.1% Higher
19.6% Higher
74.6% Higher
Analysis of growth promotion and hm-CSF
productivity kinetics showed a maximum
specific productivity (Figure 4) of
1.26 pg/cell/day. These data indicate that
increased protein expression with this cell
clone is cell-growth associated; that is, the
maximum specific productivity is seen during
log-phase growth. Additional studies analyzed
extended cell performance (Figure 5).
In the case of this clone, cells were cultured
for more than 70 days with approximately 68
cumulative population doublings (CPD). The
CPD was linear over the course of the study
indicating good stability of the cell line in this
medium. Following these evaluations, cultures
were scaled up to increase the total protein
yield by increasing the cell population density.
PF-CHO MPSq medium is a two-part powder,
protein-free medium designed for suspension
growth of CHO cell lines. The medium was
designed to be "regulatory friendly" by limiting
animal-derived components to just two, i.e.,
cod liver oil and cholesterol. This product
comes in a range of standard packaging
configurations (Table 3).
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The medium is designed to be used with a
wide range of CHO cell lines and production
processes, from straight batch to fed-batch to
continuous perfusion cultures. The formulation,
which is deficient in nucleic acids and
precursors (i.e., hypoxanthine and thymidine),
is designed for use with the dihydrofolate
reductase (DHFR) selection system and
methotrexate (MTX) induced gene amplification.
The medium is also deficient in L-glutamine
for use with the glutamine synthetase (GS)
selection system and the methionine
sulfoxamine (MSX) gene amplification system.
PF-CHO MPS can be purchased in lot sizes as
large as 400,000 L. This reduces QA/QC cost;
by reducing the number of lots needing tests.
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Some typical components that
may be added at the time of liquid
preparation are: sodium
bicarbonate, L-glutamine (for
cell clones which require this
component for growth), and
Pluronic F68e. L-glutamine
supplementation is recommended
for DHFR systems.
We anticipate that users will evaluate the
medium prior to scale-up procedures. We
recommend using cells that have been
previously adapted to the medium to eliminate
carryover variables from prior sera or media.
Table 3. Standard Packaging
Configuration
Catalog Number
SH30333.01
SH30333.02
SH30333.03
SH30333.04
SH30333.05
Through customer collaborations, we have
successfully optimized PF-CHO MPS to
specific CHO clones. Contact your sales
representative to inquire about availability
of these services.
We anticipate that users will evaluate the
medium prior to scale-up procedures. We
recommend using cells that have been
previously adapted to the medium.
References:
1. Schimke, R.T. (ed.). 1982. Gene Amplification
Coldspring Harbor Laboratory, Cold Spring
Harbor, N.Y.
2. Goeddel, D.V. (ed.). 1990. Methods in
Enzymology, Vol. 185 pp. 543-551.
Technical Information
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Pluronic, F68e is a registed trademark of BASF Corporation
Package Size
5L
10 L
50 L
100 L
1,000 L
Figure 3. Comparison of commercially available CHO medium and
PF-CHO MPSq (SH30333), using CHO 5/9 Ma 3-18 cells.
Figure 4. Cell growth performance of CHO 5/9 Ma 3-18 cells using
PF-CHO MPSq. Specific productivity appears to be strongly growthassociated with this cell clone.
Figure 5. Adaptation of cell clones to a medium often results in
decreased cell growth over multiple passages. The cell growth
performance of CHO 5/9 M! 3-18 was observed to be consistent
over an extended period of greater than 70 days when using
PF-CHO MPSq.
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