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Technical Bulletin

EX-CELL™ CD CHO: A Chemically Defined,


Animal-Component Free, Serum-Free Medium
for CHO Cells
Introduction Antibody Assay Kit
SAFC Biosciences has developed EX-CELL CD CHO, a TM • Immuno-Tek Quantitative IgG ELISA Kit, ZeptoMetrix
chemically defined, animal-component free, serum-free Corporation, Catalog No. 0801182
medium optimized for the growth of CHO-derived cell lines
producing recombinant proteins, including monoclonal Methods
antibodies (MAb). EX-CELLTM CD CHO is hydrolysate-free Media/Supplement Preparation and Storage
and contains no animal-derived components. EX-CELLTM CD CHO was supplemented with 8 mM
L-glutamine at point of use. All media were stored at
The following study was undertaken to demonstrate the 2 to 8 C protected from light. Cultures were maintained
ability of EX-CELLTM CD CHO to support the growth and MAb using aseptic technique with no antibiotic or fungicide
production in a CHO-derived (DXB11) cell line producing supplementation.
human MAb immunoglobulin G (IgG4). Additionally, the
ability of EX-CELLTM CD CHO to support the growth of a non- Culture Techniques
producing CHO-K1 cell line was assessed. Both cell lines were Prior to adaptation, CHO-K1 and DXB11 cell lines were
adapted to EX-CELLTM CD CHO using direct adaptation from maintained as suspension cultures in 125 mL shaker flasks in
serum-containing suspension cultures prior to the evaluation DMEM/F12 (CHO-K1) and IMDM (DXB11) supplemented
of growth and MAb production. with 10% FBS and 4 mM L-glutamine. After adaptation to
serum-free medium, the cells were transferred to 250 mL
Materials shaker flasks and routinely subcultured every 3 - 4 days at a
Cells density of 3 x 105 cells/mL and 4 x 105 cells/mL respectively
• DXB11, American Type Culture Collection, ATCC No. (60 mL volume per 250 mL shaker flask). The flasks were
CRL-11397 shaken on an orbital shaker at 120 - 130 rpm and were
• CHO-K1, American Type Culture Collection, ATCC No. maintained at 37 C in a humidified atmosphere with 10%
CRL-61 CO2. Cell densities and viabilities were determined by trypan
blue exclusion.
Media and Supplements
• EX-CELLTM CD Cho, with hypoxanthine and thymidine, Adaptation
SAFC Biosciences, Catalog No. 14361 The CHO-K1 and DXB11 cell lines were adapted to
• Iscove’s Modified Dulbecco’s Medium (IMDM), American EX-CELLTM CD CHO by direct adaptation. During the first
Type Culture Collection, ATCC No. 30-2005 2 - 3 passages, suspension cultures previously grown in
• Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture basal medium + 10% FBS were seeded directly into
F12 (DMEM/F12), SAFC Biosciences, Catalog No. 51448 pre-warmed EX-CELLTM CD CHO at a density of
• Fetal Bovine Serum - Gamma Irradiated (FBS), SAFC 5 x 105 cells/mL. Cells were subcultured every 3 - 4 days and
Biosciences, Catalog No. 12107 were considered fully adapted when cell densities achieved
• 200 mM L-glutamine, SAFC Biosciences, Catalog No. 59202 > 2.0 x 106 cells/mL and viabilities of > 95%.

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SAFC Biosciences, Inc. SAFC Biosciences Ltd. SAFC Biosciences Pty. Ltd.
13804 W. 107th Street Smeaton Road, West Portway 18-20 Export Drive
Lenexa, Kansas 66215 Andover, Hampshire SP10 3LF Brooklyn, Victoria 3025
USA
Phone
Toll free-USA
+1 913-469-5580
1 800-255-6032
UNITED KINGDOM
Phone
Fax
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Phone
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E-mail info-na@sial.com E-mail info-ap@sial.com
Growth Studies and Antibody Production CHO-K1 densities increase steadily over the first 4 days, reach
Growth studies were initiated after adaptation and were a plateau by day 5 and steadily decline after day 5. Viabilities
monitored over an additional 6 passages. The cells were start to drop after day 5 and are below 60% on day 7. DXB11
subcultured one additional time and daily cell counts were densities increase steadily over the first 5 days, reach a plateau
determined until culture viabilities dropped below 60%. by day 6 and decline after day 6. Viabilities start to drop
Additionally, during the last passage, daily aliquots from the after day 5 and are below 60% on day 7.
DXB11 cell suspension were taken for IgG determination.
Each aliquot was micro-centrifuged (1000 rpm) for Monoclonal Antibody Production
2 minutes, followed by collection and storage (-20 C) of the A human IgG ELISA assay kit was used to titer the production
supernatant. Antibody (human IgG) production was of IgG by the DXB11 cell line. Figure 3 depicts a typical
determined by ELISA (ZeptoMetrix Human IgG ELISA kit). viability and concurrent IgG production in the DXB11 cell line
The appropriate sample dilutions were prepared in dilution cultured in EX-CELLTM CD CHO. Viabilities remain above 90%
buffer supplied with the kit. The absorbance was read at until day 5 and proceed to decline thereafter. IgG production
405 nm on a VersaMaxTM microplate reader and calculations steadily increases with peak productivity on day 8. Additional
were performed using SoftMax® Pro. 4.0 software (both evaluations conducted by SAFC Biosciences demonstrate that
from Molecular Devices Corporation). IgG production in EX-CELLTM CD CHO is comparable to levels
typically experienced in a basal formulation + 10% FBS (Figure
Results 4). These studies demonstrate that EX-CELL TM CD CHO
supports high density cell growth and MAb production in a
Growth Studies
CHO-derived cell line.
Both the CHO-K1 and DXB11 cell lines typically reached
densities of at least 3.5 x 106 and 2.5 x 106 respectively when
subcultured every 4 days in EX-CELLTM CD CHO. Figure 1
depicts the typical growth of CHO-K1 and DXB11 cells over
the course of 6 passages in EX-CELLTM CD CHO. Figure 2
illustrates the culture longevity of CHO-K1 and DXB11 cells
over a period of 7 - 8 days in EX-CELLTM CD CHO medium.

Figure 1

CHO-K1
CHO-K1Cell Line
Cell Line DXB11 Cell Line
DXB11 Cell Line

6.00E+06
4.50E+06

4.00E+06
5.00E+06
Density (cells/mL)
(cells/mL)
(cells/mL)

3.50E+06
Density (cells/mL)

4.00E+06 3.00E+06
CellDensity

Cell Density

2.50E+06
3.00E+06
2.00E+06
Cell

Viable Cell

2.00E+06 1.50E+06
Viable
Viable

Viable

1.00E+06
1.00E+06
5.00E+05

0.00E+00 0.00E+00
1 2 3 4 5 6 1 2 3 4 5 6
Passage number
Passage number after
after adaptation
adaptation Passage
PassageNumber
numberAfter
after Adaptation
adaptation

Figure 1: Multiple passage growth of CHO-K1 and DXB11 cells in EX-CELLTM CD CHO.

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Figure 2

CHO-K1 Cell
CHO-K1 CellLine
Line DXB11
DXB11Cell LineLine
Cell

6.00E+06 120% 5.00E+06 120%


Density 4.50E+06

(cells/mL)
5.00E+06 Viability 100% 100%
4.00E+06
Density (cells/mL)

Density (cells/mL)
(cell/mL)

3.50E+06
4.00E+06 80% Density 80%
3.00E+06 Viability

CellDensity
Cell Density

Viability
Viability
Viability

Viability
3.00E+06 60% 2.50E+06 60%
2.00E+06
Viable Cell

ViableCell
2.00E+06 40% 40%
1.50E+06
Viable

Viable
1.00E+06
1.00E+06 20% 20%
5.00E+05

0.00E+00 0% 0.00E+00 0%
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8

Days
Days in
in Culture
culture Days
DaysininCulture
culture

Figure 2: Typical culture longevity of CHO-K1 and DXB11 cells in EX-CELLTM CD CHO.

Figure 3 Figure 4

1.6 120%
DXB11
1.4 Productivity
100%
1.2
Basal + 10% FBS
80%
Basal + 10% FBS
100
100
% Relative to FBS Control

1
IgG (ug/mL)
(µg/mL)

EX-CELL CDCD
CHO
% Relative to FBS Control

EX-CELL CHO
Viability
Viability

75
75
0.8 60%
IgG

0.6
40% 50
50

0.4
25
25
IgG (ug/mL) 20%
0.2 Viability
00
0
4 5 6 7 8
0%
1
Days ininCulture
Days culture

Figure 3: Human IgG production (µ µg/mL) and cell viability in Figure 4: Normalized human IgG production by DXB11 cells
DXB11 cells cultured in EX-CELLTM CD CHO. cultured in EX-CELLTM CD CHO and IMDM + 10% FBS.

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Warranty, Limitation of Remedies
SAFC Biosciences warrants to the purchaser for a period of one year from date of delivery that this product conforms to
its specifications. Other terms and conditions of this warranty are contained in SAFC Biosciences’ written warranty, a
copy of which is available upon request. ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING THE IMPLIED
WARRANTY OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE, ARE EXCLUDED. In no case will SAFC
Biosciences be liable for any special, incidental, or consequential damages arising out of this product or the use of this
product by the customer or any third party based upon breach of warranty, breach of contract, negligence, strict tort,
or any other legal theory. SAFC Biosciences expressly disclaims any warranty against claims by any third party by way of
infringement or the like. THIS PRODUCT IS INTENDED FOR PURPOSES DESCRIBED ONLY AND IS NOT INTENDED FOR
ANY HUMAN OR THERAPEUTIC USE.
Additional Terms and Conditions are contained in the product Catalog, a copy of which is available upon request.

EX-CELL™ is a trademark of SAFC Biosciences, Inc.


SoftMax® is a registered trademark of Molecular Devices Corporation.
VersaMaxTM is a trademark of Molecular Devices Corporation.

© 2006 SAFC Biosciences, Inc.

Issued March 2006 T067


0404 1105

United States Europe Asia Pacific


SAFC Biosciences, Inc. SAFC Biosciences Ltd. SAFC Biosciences Pty. Ltd.
13804 W. 107th Street Smeaton Road, West Portway 18-20 Export Drive
Lenexa, Kansas 66215 Andover, Hampshire SP10 3LF Brooklyn, Victoria 3025
USA UNITED KINGDOM AUSTRALIA

www.safcbiosciences.com Phone
Toll free-USA
Fax
+1 913-469-5580
1 800-255-6032
+1 913-469-5584
Phone
Fax
E-mail
+44 (0)1264-333311
+44 (0)1264-332412
info-eu@sial.com
Phone
Toll free-AUS
Fax
+61 (0)3-9362-4500
1 800-200-404
+61 (0)3-9315-1656
E-mail info-na@sial.com E-mail info-ap@sial.com