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Technical Bulletin

Chemically Defined CHO Feed Collection


A reference Technical Bulletin to scientific poster “Use of Chemically
Defined Supplements in Optimizing the Performance of CHO Cells”

Description Indications of Deterioration


A collection of prototype, non-cGMP, chemically defined (CD) Liquid feeds should be clear and free of particulates and
and animal-component free (ACF) feeds was developed in flocculent material. Dry powder feeds should be free flowing.
response to the need to replace undefined and regulatory- Do not use if medium is caked. Hydrated medium should be
adverse supplements traditionally used in culture of Chinese clear and free of particulates and flocculent material. Do not
Hamster Ovary (CHO) cells. The nutritional and physiological use if liquid feed is cloudy or contains precipitate. Other
requirements of CHO cells were examined using spent media evidence of deterioration may include color change, pH shift
analysis and historical data. This information was or degradation of physical or performance characteristics.
incorporated into the design of each formulation, generating
a platform of CD CHO feeds that can be used to improve cell Preparation Instructions
growth and productivity. Once the customer application- The panel of CD feed supplements is formulated without
specific formulation and feed strategy process is optimized, L-glutamine or a carbon source. For applications requiring the
SAFC Biosciences™ imMEDIAte ADVANTAGE™ and Process use of L-glutamine, supplement culture medium prior to use
and Product Services groups can determine feasibility and with the appropriate concentration of L-glutamine. SAFC
scaleability of large-scale, cGMP manufacture. Biosciences recommends L-glutamine supplementation of the
imMEDIAte ADVANTAGE non-cGMP Part No. working volume only. Glucose may be supplemented as a
81306C CD Feed 1, liquid media sterile solution directly to the feeds prior to use or can be
81307C CD Feed 2, liquid media added to cultures as a separate feed (see Feeding Protocol
81349C CD Feed 3, dry powder media below).
Preparation of CD Feed 3 (81439C)
Formulation 1. Measure 80% of final required volume of cell culture grade
water into an appropriate size mixing vessel. Water
The formulation for each CD feed is proprietary to SAFC
temperature should be 25 to 30 C.
Biosciences. CD Feeds 1 and 2 contain concentrated amino
acids, vitamins and salts. CD Feed 3 contains concentrated 2. Slowly add 21.84 g/L dry powder material while mixing.
amino acids, vitamins, salts, trace elements and lipids as well Rinse the measuring vessel with a small amount of cell
as additional supplements. All formulations are free of proteins culture grade water to remove traces of powder and add
and growth factors. to the solution. Mix for 10 minutes.
3. While mixing the solution, adjust pH to 8.8 +/- 0.1 with
Storage and Stability 1N NaOH (such as Sigma-Aldrich Product No. 415413) to
completely dissolve material.
Store medium at 2 to 8 C, protected from light. Real-time
stability studies of non-cGMP formulations have shown that 4. Adjust pH to 8.0 +/- 0.1 with concentrated HCl (such as
CD Feeds 1 and 2 are stable as liquid formulations under these Sigma-Aldrich Product No. Prod. H1758, 11.6-12.0M).
storage conditions for at least 6 months. CD Feed 3 can 5. Add cell culture grade water to the solution to bring it to
currently only be manufactured in the dry powder format. final volume and filter.
CD Feed 3 should be rehydrated prior to use and stored no
longer than 4 weeks at 2 to 8 C.

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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
+44 (0)1264-333311
+44 (0)1264-332412
AUSTRALIA
Phone
Toll free-AUS
+61 (0)3-9362-4500
1 800-200-404
www.safcbiosciences.com
Fax +1 913-469-5584 E-mail info-eu@sial.com Fax +61 (0)3-9315-1656
E-mail info-na@sial.com E-mail info-ap@sial.com
Methods for Use Product Profile
Determining Optimal Feeding Strategy Chemically defined feeds from SAFC Biosciences CD Feed
Feeding strategy should be optimized to individual cell lines, Collection were compared to undefined supplements in a
but the following general procedure can be used as a starting proprietary chemically defined SAFC Biosciences medium
point: (imMEDIAte ADVANTAGE 66659) using 5 proprietary
1. During the end of lag phase/beginning of exponential phase, recombinant IgG-producing CHO cell lines (Table 1). The
supply first feed at 10% initial culture volume (i.e. 4 mL to general feeding strategy described above was used for the
a 40 mL culture) as well as 3 g/L glucose. comparison studies.

2. During the end of exponential phase/beginning of stationary Table 1.


phase, supply second feed at 10% initial culture volume as Medium and CHO cell lines used for comparison assays
well as 3 g/L glucose. Medium Cell Line Parental
NOTE: Individualized procedure to maintain glucose can also CHO-A CHO-K1 Derived
be used. CHO-B CHO-S Derived
Other possible feeding strategies are described below: Medium Z
CHO-C CHO-S Derived
(IA 66659)
CHO-D CHO-K1 Derived
A. 10% initial culture volume on Day 2 of culture (final feed
CHO-E DHFR-
volume is 10% of initial culture volume)
B. 10% initial culture volume on Days 2, 4, 6 and 8 of culture
(final feed volume is 40% of initial culture volume)
C. 15% initial culture volume as a basal supplement on Day
0 and 10% initial culture volume (volume before Day 0
feed) on Days 3, 6 and 9 (final feed volume is 45% of initial
culture volume)
Example of feeding protocol:
For a recombinant IgG-producing CHO cell line, which
normally achieves a peak viable cell density of
4.5 - 5E6 cells/mL during a 10 - 11 day batch culture, feeding
was performed according to the procedure described below:
1. On Day 2 (viable cell density of 1.0-1.5E6 cells/mL), supply
feed at 10% initial culture volume. In addition to the CD
feed, supplement with glucose at 3 g/L from a concentrated
stock (such as Sigma-Aldrich Product No. G8769, 450 g/L)
to minimize feed volume.
2. On Day 4 (viable cell density of 3.5-4.0E6 cells/mL), supply
feed at 10% initial culture volume (i.e. 4 mL to a 40 mL
culture) and glucose at 3 g/L from a concentrated stock.

Characteristics
CD Feed 1
Osmolality (as supplied)
200 +/- 20 mOsm/kg H20
pH (as supplied)
7.0 +/- 0.3

CD Feed 2
Osmolality (as supplied)
250 +/- 20 mOsm/kg H20
pH (as supplied)
7.0 +/- 0.3

CD Feed 3
Osmolality
(as prepared; dependent on pH during preparation)
290 +/- 20 mOsm/kg H20
pH (as prepared)
8.2 +/- 0.3
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Results

Figure 1

Growth and Productivity of CHO-K1 Derived Cell Lines in Medium Z


1800 4.50E+07

1600 4.00E+07
Peak Volumetric Productivity

1400 3.50E+07

Viable cells per mL*day


1200 3.00E+07
IgG mg/L

1000 2.50E+07

800 2.00E+07

600 1.50E+07

400 1.00E+07

200 5.00E+06

0 0.00E+00
Glucose

CD Feed 1

CD Feed 2

CD Feed 3

Glucose

CD Feed 1

CD Feed 2

CD Feed 3
Soy

Soy
Wheat

Wheat
Yeast

Yeast
CHO-A CHO-D

Figure 2

Growth and Productivity of CHO-S Derived Cell Lines in Medium Z

800 4.50E+07

700 4.00E+07
Peak Volumetric Productivity

3.50E+07

Viable cells per mL*day


600
3.00E+07
500
IgG mg/L

2.50E+07
400
2.00E+07
300
1.50E+07
200
1.00E+07
100 5.00E+06

0 0.00E+00
Glucose

CD Feed 1

CD Feed 2

CD Feed 3

Glucose

CD Feed 1

CD Feed 2

CD Feed 3
Soy

Soy
Wheat

Wheat
Yeast

Yeast

CHO-B CHO-C

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Figure 3

Growth and Productivity of DHFR- Cell Line in Medium Z


45 4.50E+07

40 4.00E+07
Peak Volumetric Productivity

35 3.50E+07

Viable cells per mL*day


30 3.00E+07
IgG mg/L

25 2.50E+07

20 2.00E+07

15 1.50E+07

10 1.00E+07

5 5.00E+06

0 0.00E+00
Glucose

CD Feed 1

CD Feed 2

CD Feed 3
Soy

Wheat
Yeast

CHO-E

Conclusions
• In the studies shown, we observed a significant increase in
cell growth and up to a 150% increase in recombinant
protein production when multiple CHO cell lines were fed
with any of the three supplements in the CD CHO Feed
Collection as compared to feeding with only glucose. When
these same cell lines were tested in other proprietary CD
media, increases in protein production of up to 300% were
observed (data not shown).
• Feeding with chemically defined supplements achieves similar
cell growth and productivity as feeding with undefined
supplements and can improve specific productivity, as
observed in most of the tested cell lines (data not shown).
• Responsiveness to feeding with individual undefined
supplements and CD feeds is dependent on cell line and
culture medium. Awareness of this is important for the
development of platform systems.

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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.

SAFC Biosciences™, the SAFC Biosciences logo and imMEDIAte ADVANTAGETM are
trademarks of Sigma-Aldrich Biotechnology L.P.

© 2008 SAFC Biosciences, Inc.

Issued October 2008 T102

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