0

1

2

3

Tite

r (hI

gG) g

/L

PEI MAX(No enhancer or feed)

ExpiFectamine CHO(With enhancer and feed)

Days of culture

Tite

r (h

IgG

) g/L

0 1 2 3 4 5 6 7 80.0

0.5

1.0

1.5

10x106 cells/mL

6x106 cells/mL

Days of culture

Tite

r (h

IgG

) g/

L

0 1 2 3 4 5 6 7 80.0

0.5

1.0

1.5

2.0

10x106 cells/mL

6x106 cells/mL

Chao Yan Liu, Virginia Spencer, Shyam Kumar, Jian Liu, Ping Liu, Sara Barnes, Henry Chiou, Jonathan F. Zmuda. Thermo Fisher Scientific Inc.

Figure 3. Characterization of ExpiCHO-S cells. (A) ExpiCHO-S cells. (B) Stability of protein expression over 18 passages. (C) Growth and viability curves for ExpiCHO-S cells grown in standard shake flask culture.

Abstract and introduction 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 post-translational 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 only 1% to 2% of 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 capable of generating g/L 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.

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 medium 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. Acknowledgments 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.

ExpiCHO system: surpassing the performance of Expi293 in a transient CHO expression system

Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • thermofisher.com

Figure 5. Characteristics of ExpiFectamine CHO Transfection Reagent (A) When used in conjunction with ExpiCHO enhancer and feed, ExpiFectamine CHO Transfection Reagent 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 0.6 µg/mL of culture volume generate maximal protein titers, corresponding to half of the industry standard of 1.0 µg/mL plasmid DNA.

II. ExpiCHOTM Expression Medium and ExpiCHOTM Feed

VI. Kinetics of protein production

Figure 6. ExpiCHO protocol(s).

Figure 1. Systems-based approach to increased transient protein expression

I. Generation of high-expressing ExpiCHO-STM cells

Figure 10. Protein quality and glycosylation patterns in ExpiCHO and Expi293TM systems

(A) SDS-PAGE of human IgG, under non-reducing (left) and reducing (right) conditions. (B) Size exclusion chromatography of human IgG. (C) Human IgG glycans.

Figure 4. Optimization of ExpiFectamine CHO Enhancer and ExpiCHO Feed addition

(A) Protein titers are reduced by 50% or more without the addition of the ExpiFectamine CHO enhancer reagent. (B) Two equal volume feeds on Days 1 and 5 post-transfection double protein titers.

Figure 2. Workflow for identifying high-expressing CHO clones. CHO cells were transiently transfected and evaluated for protein expression using the Molecular Devices ClonePixTM System. Selected clones were further evaluated via transfection with plasmids for multiple proteins.

Figure 7. Kinetics of hIgG expression, viability, and viable cell density. (A) 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 32°C. Red line: Max titer protocol consisting of two feeds and temperature shift to 32°C. Insert: Viable cell density post-transfection. (B) Viability post- transfection for ideal expression run. (C) Viability post-transfection for troubleshooting (red line).

V. Workflow

III. ExpiFectamineTM CHO Transfection Enhancer and ExpiCHO Feed

ExpiCHO Expression Medium attributes •  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 •  Manufactured under cGMP •  Supports high-density cell growth •  Matched to a specific feed •  Free from regulatory/import/export limitations

ExpiCHO-S cell line attributes • Derived from GMP CHO-S cells • Adapted for high-density culture • High specific productivity (~27 pcd) • No clumping during expression runs • Short doubling time (~17 hours) • Stable growth and expression over 20+ passages

B

A

C

A B

B

Figure 9. Scalability of the ExpiCHO system

IV. ExpiFectamineTM CHO Transfection Reagent A

VIII. ExpiCHO system scalability

X. Protein characterization

B A

C

A

B

C

B A

Viability

Final volume 3.5 mL Shake speed 225 rpm

(A) The ExpiCHO system is directly scalable from 125 mL to 2 L flask sizes; 3 L flasks require reduction in shake speed to 70 rpm. (B) High-titer protocol in 24 deep well plates with shake speed of 225 rpm.

IX. Expression of selected proteins in the ExpiCHO system

Table 1. Titer comparison in current and ExpiCHO systems 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 systems are shown. IgG titers in ExpiCHO system range from 0.6–97 folds (Avg. 5 fold) than those obtained using various other systems. Additionally, 7 proteins from this panel were not expressed in Expi293 cells yet expressed at high levels in the ExpiCHO system.

VII. System flexibility: ultrahigh-density transfection

Figure 8. Ultrahigh-density transfection for rapid protein expression Ultrahigh-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.

B A Standard protocol High-titer protocol

Expi293 system

Expi293 system

Tite

r (hI

gG) g

/L

One Two 0

1

2

3

ExpiCHO feed

0.2 0.4 0.6 0.8 1.0 1.20

1

2

3

Tite

r (hI

gG) g

/L

DNA concentration (µg/mL)

30-fold

Days of culture

Tite

r (hI

gG) g

/L

0 1 2 3 4 5 6 7 8 9 10 11 12 13 140

1

2

3

125mL 250mL 500mL 1L 2L 3L0

1

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4

Tite

r (hI

gG) g

/L

115 - 125 rpm 60 - 70 rpm

25 mL 50 mL 100 mL 200 mL 400 mL 750 mL

Flask size

Transfection volume

Shake speed

35 mL 70 mL 140 mL 280 mL 560 mL 1000 mLFinal volume

Protein

Expi293  or  current  system  (mg/L)

ExpiCHO        (mg/L)

Fold  increase

Protein

Expi293  or  current  system  (mg/L)

ExpiCHO        (mg/L)

Fold  increase

Protein

Expi293  or  current  system  (mg/L)

ExpiCHO        (mg/L)

Fold  increase

Human Ab 26.6 2580 97 Human Ab Undetectable 500 Murine Ab 60 240 4.04 147 33 Precipitated 330 60 240 4.04 56 14.6 Rabbit Ab 5 120 20 76 196 2.6

22 266 12.4 17 178 11 12 18 1.548 334 6.9 420 1733 4.1 16 17 1.136 190 5.3 271 1143 4.2 119 48 0.418 78 4.3 196 698 3.6 135 47 0.385 314 3.7 307 966 3.2 Bi-TE Western blot Western blot 25

1025 3250 3.2 182 521 2.9 Bi-specific 28 117 4.2170 410 2.4 411 1141 2.8 26 90 3.5138 325 2.4 385 1065 2.8 126 250 2.025 50 2.0 481 1249 2.6 NA 763102 204 2.0 199 500 2.5 NA 66057 92 1.6 454 1100 2.4 Secreted 216 1412 6.5336 491 1.5 568 1377 2.4 Non-Ab 15 82 5.5565 864 1.5 393 942 2.4 Western blot Western blot 5.0450 600 1.3 125 284 2.3 164 68 0.4350 450 1.3 558 1202 2.2 NA 206309 383 1.2 702 1402 2.0 Fab 85 366 4.3243 276 1.1 549 983 1.8 10 10 1.0352 371 1.1 195 306 1.6 FC-fusion 5 8 1.7565 620 1.1 452 658 1.5 His-tag HLA Undetectable 12 12309 302 1.0 215 273 1.3 Viral protein Western blot Western blot 3-5139 129 0.9 Undetectable 79 Cytokine Western blot Western blot 5-1011 9 0.8 Murine Ab 216 1412 6.5 FcR Western blot Western blot 2-3565 365 0.6 20 86 4.3 EPO 252 568 2.3

For Research Use Only. Not for use in diagnostic procedures. © 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

Non-reduced purified IgG Reduced purified IgG

ExpiCHO Standard protocol

ExpiCHO High titer protocol

ExpiCHO Max titer protocol

Stable CHO-S Expi293 system

86% 92% 91%

95% 92%

Days of culture

Expi293 system Standard protocol § 37ºC § Enhancer § 1 feed

High titer protocol § 32ºC § Enhancer § 1 feed

Max titer protocol § 32ºC § Enhancer § 2 feeds

Viable cell density

3 10 180

1

2

3

Tite

r (hI

gG) g

/L

Passage number Days of culture

VC

D (x

106

cells

/mL)

(sol

id li

nes) %

Viability

(dotted lines)

0 1 2 3 4 5 6 70

5

10

15

20

25

0

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100

Passage 10

Passage 34

Days of culture

Tite

r (hI

gG) g

/L

0 1 2 3 4 5 6 7 8 9 10 110.0

0.5

1.0

1.5

2.0

Control 125 mL flask High-titer protocol

Tite

r (hI

gG) g

/LExpiFectamine CHO enhancer

0

1

2

3

4

_ +