Streamlined Strategy for Rapid Development and Optimization of Cell Culture Media

Supporting High Recombinant Protein Production in Chinese Hamster Ovary Cells

Min Zhang, Avril Lawshé, Kerry Koskie, Terrell Johnson, Matthew V. Caple, and James S. Ross
Cell Sciences & Development, SAFC Biosciences, Saint Louis, Missouri 63103

Introduction A B
Comb P C E D
1 0.000 0.500 0.500 0.000
The robustness of Chinese Hamster Ovary (CHO) cells in large-scale recombinant protein production P 2 0.000 0.666 0.167 0.167
3 0.167 0.167 0.666 0.000

and their ability to sustain active biological function of expressed proteins prompts their wide usage in 4
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pharmaceutical companies. However, recombinant CHO cells are challenging to optimize because of the 6
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0.500
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0.500
0.167

diverse nutritional needs unique to every clone. 8

9
0.500
0.334
0.000
0.000
0.000
0.333
0.500
0.333
10 0.167 0.000 0.167 0.666

It is essential to develop the most efficient and optimal media development strategy possible. The 11
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traditional approach frequently used in the industrial setting is to test one component at a time to 13
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determine its optimal level, which is labor intensive, costly and time consuming. SAFC Biosciences’ CHO a b
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0.666
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0.167
0.500
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0.000
0.000
17 0.167 0.167 0.000 0.666
cell culture platform includes high-throughput screening technology, a diverse selection of CHO media 18 0.000 0.334 0.333 0.333
19 0.000 0.000 0.000 1.000
(both animal-component free and chemically defined formulations), state-of-the art cell engineering (c)
20
21
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0.666
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0.167
technology and statistical Design of Experiment (DOE) design software; the platform allows us to make 22
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CHO media optimization an extremely efficient process. 24

25
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0.666
0.500
0.000
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0.167
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0.167
C (d) E 26 0.250 0.250 0.250 0.250
In this presentation, we will introduce the new streamlined strategic approach to speed media 27
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development by applying the “CHO Media Library”, DOE media mixing screening, spent medium D 29
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component analysis and bioprocess cell feeding strategies. With this strategy, compared to the
traditional method, we can analyze multiple criteria simultaneously to determine synergistic responses.
We can then identify one or more media mixtures that provide the desired user outcomes. Meanwhile,
an example will be used to depict use of this strategy to efficiently develop optimized formulations for a
humanized IgG-producing CHO clone.
Materials and Methods
Cell lines and media:
Figure 2. DOE “Pyramid” Design
A) Schematic representation of DOE design. Four SAFC Biosciences’ formulations (C, D, E and P) have been
chosen for DOE mixing analysis. To simplify the experimental design, only the combinations on the surfaces
(a~d) of pyramid have been evaluated, including vertices points (red spots), centers of edges (orange
spots), surface centroids (yellow spots), axial check blends (green spots) and overall centroid (big red spot)
B) DOE combination table (including 26 combinations and 4 replicates of overall centroids).
A CHO Media Library Screening B DOE Screening
1000 1000
C Mix #9

IgG Productivity (mg/L)

Table1: shows the CHO cell lines tested with CHO Media Library. The stock cultures of these cells were cultured in 900 Mix #16

IgG Productivity (mg/L)

900 D
E Mix #25
800 CM1
EX-CELL™ CHO DHFR- Medium, an animal-component free formulation (Item No. C8862 or proprietary formulations). 800 P
CM1
700 CM2 700
The stock cultures were pre-adapted to the test formulations or directly seeded into the test formulations without 600
600
adaptation. All SAFC Biosciences formulations and two competitor media were supplemented with 6 mM L-Glutamine 500 500
unless otherwise specified. The data for this presentation was from 125 mL shaker flask culture. 400 400
300 300
200 200
Cell Line Cell Line Lineage Recombinant Protein Produced 100 100
0 0
CHO-K1 CHO-K1 parental None 5 6 7 8 9 10 11 12 5 6 7 8 9 10 11

CHO-S CHO-S parental None Culture Time (days) Culture Time (days)

CHO-AP1 CHO-K1 derived Alkaline Phosphatase Figure 3. IgG Productivity Comparison

CHO-AP2 CHO-K1 derived Alkaline Phosphatase A) IgG production in selected formulations from CHO Media Library along with 2 competitor media.
CHO-hGH1 CHO-K1 derived Human Growth Hormone B) IgG production in selected mixing formulations after DOE screening along with competitor medium A.
CHO-hGH2 CHO-K1 derived Human Growth Hormone DOE mixing dramatically improved the IgG productivity ~ 2-fold higher than that in competitor medium A.
Recombinant CHO line 1 CHO-K1 derived Human IgG
Recombinant CHO line 2 CHO-S derived Human IgG

Table 1. CHO Cell Line Models for CHO Media Library Study
Analytical methods and productivity assays:
Alkaline phosphatase activity: alkaline phosphatase activity was measured with Alkaline Phosphatase
Fluorescence Detection Kit according to the manufacturer’s protocol (Sigma-Aldrich, AP-F).
Human growth hormone: hGH productivity was quantified with hGH ELISA Detection Kit (Roche). The
procedures were conducted at the manufacturer’s protocol with minor modifications.
Human IgG: IgG concentration was measured with Protein A affinity chromatography.
Statistical analysis:
In the DOE mixing experiment, User-Defined Design category was used to generate the mixing
combinations to make a “Pyramid” model (Figure 2). The DOE screening data was analyzed by Design-
Expert® Version 7.0.1 (Stat-Ease). Figure 4. DOE Statistical Analysis and Numerical Optimization
A) DOE optimization based on the cell growth (ICA: specific cell growth). It can be observed that more of
formulation C would be beneficial to improve cell growth.
Results and Discussion
B) DOE optimization based on IgG productivity. More of formulation P would be beneficial to improve IgG
production. Therefore, based on the features of the specific cell lines, the different mixing strategies will be
Genomics/Proteomics/ applied to fit the needs.
Metabolomics
A Viable Cell Density B D-Glucose
High-throughput 5.00E+06 7
Mix #25 (37) 2
screening with 4.50E+06 Mix #25 (31) 6
1.8
1.6
CM1 (37)
multiple CHO lines 4.00E+06 CM1 (31)
1.4
(g/L)
-Gluc (g/L)

3.50E+06 5 1.2
Cells/mL

1
L-Gluc

0.8
3.00E+06 4
Customer CHO
g/L

0.6
L

2.50E+06 0.4

cell lines 2.00E+06

3 0.2
0
37 31
SAFCB wide CHO Top CHO Media 1.50E+06 2 Mix #25 (37)
1.00E+06 Mix #25 (31)
formulation formulations Library 5.00E+05
1 CM1 (37)
CM1 (31)
selections 0.00E+00 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 0 1 2 3 4 5 6 7 8 9 10 1112 1314 1516 1718

Culture Time (days) Culture Time (days)

Top 3-5 C D IgG Productivity

Viable Cell Density
formulations 6.00E+06 1200
1100
IgG (mg/L)

5.00E+06 1000
900
4.00E+06
Cells/mL

800
700
3.00E+06 600
500
2.00E+06 400
Mix #25 (37) 300 Mix #25 (37)
1.00E+06 Mix #25 (37+Gluc) 200 Mix #25 (37+Gluc)
Pharmaceutical Culture Best DOE mixing Mix #25 (31) 100
Mix #25 (31)
Mix #25 (31+Gluc) Mix #25 (31+Gluc)
protein optimization formulations screening 0.00E+00 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 7 8 9 10 11 12 13 14 15 16 17 18
production
Culture Time (days) Culture Time (days)

Figure 1. Schematic representation of the strategic platform for CHO media development
To develop the CHO Media Library, multiple CHO cell lines were tested against a wide selection of CHO
formulations from SAFC Biosciences. Top formulations were ranked based on cell growth and protein
productivity. Customer CHO cell lines will be screened with the CHO Media Library and the top 3 – 5
formulations will be selected for DOE mixing screen to identify the best formulation mixtures supporting
the customer cell lines. After optimization of cell culture conditions (e.g. temperature shift and cell
feeding), the selected formulations will significantly improve the pharmaceutical protein production in
an efficient manner. Meanwhile, the selected top formulations and SAFC Biosciences’ ongoing state-
of-the-art biology studies (Genomics/Proteomics/Metabolomics) will continue enriching the contents of
CHO Media Library.
02954-661311
Figure 5. Cell culture condition optimization with Mix #25
A) The cell growth under temperature shift study. The cell cultures were switched from 37 °C to 31 °C
on day 6.
B) L-glucose consumption rate has been significantly slowed down in 31 °C cultures. The inset compares
the L-glucose level in the Mix #25 with the two temperatures on day 9
C) The cell growth under the temperature shift and L-glucose feeding conditions in the Mix #25
D) IgG productivity in Mix #25 under the temperature shift and L-glucose feeding condition.
Conclusions
• SAFC Biosciences’ cell culture platform (high-throughput screening, diverse selections of CHO media, cell
line engineering, DOE design and systems biology methodologies) facilitates the new strategic approach
to speed media development. In comparison with traditional development approaches, use of this
platform dramatically reduces costs and development time (usually less than 5 months).
• In this study, we first present the four-component DOE analysis (pyramid model) which turns out to be
more informative and efficient than the conventional three-component DOE analysis (triangle model).
• With an IgG-producing CHO clone, the CHO Media Library platform has developed several well-
performing formulations supporting IgG production, Mix 25 demonstrates the efficacy of this platform by
nearly tripling the IgG productivity as compared to the control (competitor A) after culture modification
(temperature shift).