Surpassing the Performance of 293 in a Transient CHO Expression

ExpiCHO™: Surpassing the Performance of 293 in a Transient CHO Expression System
Chao Yan Liu, Virginia Spencer, Shyam Kumar, Jian Liu, Ping Liu, Sara Barnes, Henry Chiou*, Jonathan F. Zmuda. Thermo Fisher Scientific, 7335 Executive Way, Frederick MD, *5791 Van Allen Way,
Carlsbad, CA.
V. Workflow
ABSTRACT & INTRODUCTION
A
Protein
•  Adapted for high-density culture
•  High specific productivity (~27pcd)
•  No clumping during expression runs
•  Short doubling time (~17 hours)
•  Stable growth and expression over
20+passages
Human Ab
B
C
97
33
14.6
12.4
6.9
5.3
4.3
3.7
2.4
2.4
2.0
2.0
1.6
1.5
1.5
1.3
1.3
1.2
1.1
Human Ab
352
565
309
139
11
565
Undetectable
Precipitated
5
17
216
20
60
60
76
12
16
119
135
Rabbit Ab
Murine Ab
371
620
302
129
9
365
500
330
120
178
1412
86
240
240
196
18
17
48
47
1.1
1.1
1.0
0.9
0.8
0.6
Bi-TE
Bi-Specific
Western Blot
28
26
126
NA
NA
Secreted
216
Non-Ab
15
Western Blot
164
NA
Fab
85
10
FC-Fusion
5
His Tag HLA Undetectable
Viral protein Western Blot
Cytokine
Western Blot
FcR
Western Blot
20
11
6.5
4.3
4.0
4.0
2.6
1.5
1.1
0.4
0.3
Western Blot
117
90
250
763
660
1412
82
Western Blot
68
206
366
10
8
12
Western Blot
Western Blot
Western Blot
25
4.2
3.5
2.0
6.5
5.5
5.0
0.4
4.3
1.0
1.7
12
3-5
5-10
2-3
One
0.030
ExpiCHO Feed
10x10 /mL
6
6x10 /mL
0.5
1
2
3
4
5
Days post transfection
6
6.00
8.00
10.00
12.00
14.00
16.00
2
1
0
0.4
0.6
0.8
1.0
AU
4.00
6.00
8.00
(A) When used in conjunction with ExpiCHO Feed and Enhancer, ExpiFectamineCHO generates greater than
30-fold higher titers than PEI alone. (B) Despite the high density of cells at the time of transfection, plasmid
DNA levels as low as 0.5 µg/mL of culture volume generate maximal protein titers, corresponding to half of the
industry standard of 1.0 µg/mL plasmid DNA.
10.00
12.00
14.00
6x106/mL
8.853
95%
Stable CHO-S
1
2
3
4
5
Days post transfection
6
20.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
Minutes
92%
AU
10.597
7.279
7.659
6.069
0.020
0.010
0.000
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
0.00
20.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
Minutes
ExpiCHO
Stable CHO-S
Expi293
58%
2% 1%
0
18.00
0.00
Expi293
0.040
0.030
0.010
C
16.00
Minutes
0.050
82%
87%
7% 3%
20% 9%
7
Figure 10. Protein Quality and Glycosylation Patterns in ExpiCHO and Expi293
(A) Human IgG SDS PAGE (Left) Non-Reduced and (Right) Reduced purified IgG. (B) Human IgG SEC. (C)
Human IgG Glycans.
CONCLUSIONS
We describe a systems-based approach for enhancing levels of transient protein production in
CHO cells that allows for the production of recombinant proteins at levels exceeding those of
the Expi293 system while maintaining activity, purity and glycosylation patterns comparable to
those observed in stably transfected CHO-S cells. This performance enhancement was made
possible through the incorporation of multiple novel reagents including: (1) a high-expressing
CHO cell clone, (2) a CD/AOF culture media that allows for high density CHO growth and
transfection, (3) an optimized CHO cell transfection reagent, (4) a novel CHO feed optimized
for transient transfection culture conditions, (5) a post-transfection enhancer solution and (6) a
simple to perform workflow.
1.5
1.0
0.5
0
1
2
3
4
5
6
7
8
Days of Culture
Control 125 mL Flask
Figure 5. Characteristics of ExpiFectamineCHO Transfection Reagent
10.651
6.816
7.232
7.633
20.00
2.00
AU
Expi293
0.0
1.2
DNA Concentration (µg/mL)
0.010
Minutes
1
91%
0.000
18.00
0.00
0.020
10x106/mL
ExpiCHO
Max Titer Protocol
8.855
4.00
0.040
2.0
B
0.030
6.117
2.00
Minutes
Ultra-high density transfection can generate protein titers in half the time. (A) (Red line) Transfection of ExpiCHO-S
cells at density of 6x106 cells/mL using the Standard protocol. (Blue line) High density transfection of ExpiCHO-S
cells at 10x106 cells/mL. (B) (Red line) Transfection of ExpiCHO cells at density of 6x106 cells/mL using the High
Titer protocol. (Blue line) High density transfection at 10x106 cells/mL
A
0.040
0.000
0.00
0
7
hIgG Titer (g/L)
hIgG Titer (g/L)
6
92%
0.020
0.010
0.050
VIII. ExpiCHO System Scalability
3
0.2
High Titer Protocol
0.050
ExpiCHO
High Titer Protocol
0.020
10.663
0.010
86%
0.040
AU
0.020
Max Stable Expi293
8.848
8.854
0.050
ExpiCHO
Standard Protocol
11.963
0.030
0.00
Figure 8. Ultra-High Density Transfection for Rapid Protein Expression
Two
IV. ExpiFectamineCHO Transfection Reagent
hIgG Titer (g/L)
1.0
0
(A) Protein titers are reduced by 50% or more without the addition of the ExpiCHO Enhancer reagent. (B) Two
equal volume feeds on Days 1 and 5 post-transfection double protein titers.
(With Enhancer and Feed)
Expi293
0.0
Figure 4. Optimization of ExpiCHO Enhancer and Feed Addition
(No Enhancer or Feed)
2
B
Standard Protocol
hIgG Titer (g/L)
hIgG Titer (g/L)
hIgG Titer (g/L)
hIgG Titer (g/L)
1
0
3
1.5
A
2
+
ExpiFectamineCHO
0.040
0.000
ExpiCHO Enhancer
PEI Max
0.050
0.030
3
1
0
B
VII. System Flexibility: Ultra-High Density Transfection
2
1
High
Reduced purified IgG
0.000
3
30 fold
Std
ExpiCHO
7.450
7.635
(Green line) Standard Protocol consisting of one feed and no temperature shift. (Blue line) High Titer Protocol
consisting of one feed and temperature shift to 32ºC. (Red line) hIgG kinetics using the Max Titer Protocol
consisting of two feeds and temperature shift to 32ºC. (B) Viability post transfection. (C) Viable cell density post
transfection for Standard and Max Titer protocols.
6.110
(A)
B
2
Max Stable Expi293
Non-Reduced purified IgG
Figure 7. Kinetics of hIgG Expression, Viability and Viable Cell Density
4
_
High
ExpiCHO
III. ExpiCHO Expression Medium and ExpiCHO Feed
0
Std
A
•  Manufactured under cGMP
•  Supports high-density cell growth
•  Matched to a specific feed
•  Free from regulatory/import/export limitations
A
X. Protein Characterization
10.621
Viable Cell Density
8.850
C
Viability
7.240
7.637
B
Titer
6.113
A
11.965
VI. Kinetics of Protein Production
10.640
(A) ExpiCHO-S cells. (B) Stability of protein expression over 18 passages. (C) Growth curve for ExpiCHO-S cells
grown in standard shake flask culture.
7.247
7.664
Figure 3. Characterization of ExpiCHO-S Cells
•  No supplementation required
•  One medium for growth and transfection
•  Formulated specifically for transient transfection
•  Chemically-defined (CD)
•  Animal origin-free (AOF)
•  Serum-free
•  Protein-free
CHO cells were transiently transfected and evaluated for protein expression using the ClonePix system
(Molecular Devices). Selected clones were further evaluated via transfection with plasmids for multiple
proteins. Control protein titers using an existing CHO clone as well as Control titers for the pre-clonal pool.
2580
147
56
266
334
190
78
314
410
325
50
204
92
491
864
600
450
383
276
ExpiCHO Fold (mg/L)
Increase
Expression levels of human IgG, Rabbit IgG, Murine IgG and a broad range of non-Ab proteins in Expi293 or
other transient expression systems and ExpiCHO system are shown. ExpiCHO IgG titers range from 0.6-97x
(Avg. 5x) those obtained using various other systems. Additionally, 6 proteins from this panel were nonexpressors in 293 cells yet expressed at high levels in ExpiCHO.
ExpiCHO Expression Media Attributes
Figure 2. Workflow for Identifying High-Expressing CHO Clones
26.6
4
4
22
48
36
18
85
170
138
25
102
57
336
565
450
350
309
243
Expi293 or Current System (mg/L)
Protein
Table 1. Titer Comparison in Expi293 or Current and ExpiCHO systems
Figure 6. ExpiCHO Protocol(s)
II. ExpiCHO Expression Medium and ExpiCHO Feed
Figure 1. Systems-Based Approach to Increased Transient Protein Expression
Expi293 or Expi293 or Current ExpiCHO Fold ExpiCHO Fold Protein
Current System (mg/L) Increase
(mg/L) Increase
System (mg/L)
(mg/L)
AU
CHO cells are the predominant host for biotherapeutic protein expression, with roughly
70% of licensed biologics manufactured in CHO. Multiple attributes make CHO cells
desirable for bioproduction including the ability to adapt to high-density suspension
culture in serum-free and chemically-defined media and the incorporation of posttranslational modifications that are biologically-active in humans. For these reasons, the
ability to produce transient CHO-derived proteins early on during drug development is
highly advantageous to minimize, as much as possible, changes in protein quality/
function observed when moving from R&D to bioproduction. Unfortunately, CHO cells
express lower levels of protein than HEK293 cells in existing transient systems, in some
instances 50-100 times less than the best 293-based systems, and only modest titer
improvements are obtained through the optimization of individual components of existing
transient CHO workflows. To address the significant unmet need for higher transient
CHO protein titers, systems-based approaches were employed whereby the latest
advances in cell culture media, feeds, transfection reagents and expression enhancers
were optimized in conjunction with a new high-expressing CHO cell clone to generate a
simple and robust workflow for transient protein expression in CHO cells capable of
generating gram per liter protein titers in 10-14 days. These advances will allow for
unprecedented access to CHO-derived proteins early on during candidate selection and
may serve to revolutionize the use of CHO cells for transient protein expression during
the drug development process.
ExpiCHO-S Cell Line Attributes
•  Derived from GMP CHO-S cells
IX. Expression of Select Proteins in ExpiCHO System
9
10
11
High Titer Protocol
ACKNOWLEDGEMENTS
We would like to thank Michael Gillmeister for performing the glycan analysis on the human
IgG samples and Brian Paszkiet for assisting with the transfection efficiency studies.
Figure 9. Scalability of the ExpiCHO System
(A) ExpiCHO is directly scalable from 125mL to 2L flask sizes, 3L flasks require reduction in shake speed to 70rpm.
(B) High Titer protocol in 24 deep well plates with shake speed of 225rpm.
Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com
18.00