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hIgG Titer (g/L)
0
1
2
3
4
ExpiFectamine CHO(with Enhancer and Feed)
PEI(no Enhancer or Feed)
30-fold
TIter (m
g/L)
Day 3
Day 5
Day 7
Roland Leathers, Chao Yan Liu, Virginia Spencer, Shyam Kumar, Jian Liu, Ping Liu, Henry Chiou*, Jonathan F. ZmudaThermo Fisher Scientific, 7335 Executive Way, Frederick MD, *5791 Van Allen Way, Carlsbad, CA.
Figure 3. Characterization ofExpiCHO-SCells(A) ExpiCHO-S cells. (B) Stability of protein exp ression ov er 18 pass ages. (C ) Growth c urve fo rExpiCHO-S cells grown in standard shake flask culture.
ABSTRACT & INTRODUCTIONCHO cells are the predominanthost for biotherapeutic protein expression,with roughly70% of licensed biologics manufactured in CHO. Multiple attributes make CHO cellsdesirable for bioproduction including the ability to adapt to high-density suspensionculture in serum-free and chemically-defined media and the incorporation of post-translational modifications thatare biologically-active in humans. For these reasons,the ability to produce transientCHO-derived proteins early on during drug developmentis highly advantageous to minimize, as much as possible, changes in proteinquality/function observedwhen moving fromR&D to bioproduction. Unfortunately,CHOcells express lower levels ofprotein than HEK293 cells in existing transientsystems, insome instances 50-100 times less than the best293-based systems,and only modesttiter improvements are obtained through the optimization of indiv idual components ofexisting transientCHO workflows. To address the significant unmet need for highertransientCHO protein titers,systems-based approaches were employed whereby thelatest advances in cell culture media, feeds, transfection reagents and expressionenhancers were optimized in conjunction with a new high-expressing CHOcell c lone togenerate a simple and robust workflow for transient protein expression in CHO cellscapable ofgenerating gramper liter protein titers in 10-14 days. These advances willallow for unprecedented access to CHO-derived proteins early on during candidateselection and may serve to revolutionize the use of CHO cells for transient proteinexpressionduring thedrugdevelopmentprocess.
CONCLUSIONSWe describe a systems-based approach for enhancing levels of transient proteinproduction in CHO cells that allows for the production of recombinant proteins at levelsexceeding those of the Expi293 systemwhile maintaining activ ity,purityand glycosylationpatterns comparable to those observed in stably transfected CHO-S cells. Thisperformance enhancementwas made possible through the incorporation ofmultiple novelreagents including: (1) a high-expressing CHO cell c lone, (2) a CD/AOF culture mediathat allows for high density CHO growth and transfection, (3) an optimized CHO celltransfection reagent, (4) a novel CHO feed optimized for transient transfection cultureconditions, (5)a post-transfection enhancer solution and (6) a simple to performworkflow.
ACKNOWLEDGEMENTSWe would like to thank Michael Gillmeister for performing the glycan analysis on thehuman IgG samples and Brian Paszkiet for assisting with the transfection effic iencystudies.
ExpiCHO™: Surpassing the Performance of 293 in a Transient CHO Expression System
Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • lifetechnologies.com
Figure 5. Characteristics ofExpiFectamineCHO TransfectionReagent(Top panel) Ti me c ourse of GFP expr ession in ExpiCHO-S c ells. Insets show flow cytometry data fo rcorresp onding cultu res. (A) When used in conjunc tion with ExpiCHO Feed and Enhancer ,ExpiFectamineCHO g ener ates gre ater th an 30-f old highe r titers tha n PEI alone. (B) Despite the hig hdensity of cells at the time of t ransfecti on, plasmid DNA levels as low as 0.43 µg/ mL of final cultur evolume gene rate maximal p rotein titers , co rresp onding to less than half of the indust ry sta ndar d o f 1. 0µg/mL plasmid DNA.
Figure 6. Identification of OptimalPost-Transfection Enhancers(A) Combinatio ns of differe nt expr ession enh ancers we re tested by multi-fact orial DOE for effects o nprotein titers. Data f rom a sub -set o f enh ancer combi nations are s hown, in dicating t he impact of vario usenhance r formulations o n pro tein titers . (B) Protein tit ers ar e reduc ed by 50% or more wit hout th eaddition of the ExpiFectamineCHO Enhancer reagent.
II. ExpiCHO Expression Medium and ExpiCHO Feed
III. ExpiFectamineCHO Transfection Reagent
VI. Kinetics of Protein Production
Figure 7.Transient Transfection Protocol– 7 or 8Stepsto Protein Production
Figure 1. Systems-BasedApproach toIncreasedTransientProtein Expression
I. Generation of High-Expressing ExpiCHO-S CellsTransfection of CHO cells with
Protein X
Selection of high expressing clones by
Clone Pix
Clone expansion into 6 well dishes
Screen clones for Protein Y expression
Clone expansion into 30 mL shake
flasks
Screen clones for Protein Z expression
Master Cell Bank Generation
Figure 11. Glycosylation Patterns in CHO (Transient and Stable)andHEK293Human IgG sup erna tant samples were collect ed and p urified usin g POROS® MabCaptu re® A resin .Following PNGase digestio n and APTS labeling, glyca n profiles we re analyz ed on an Applie dBiosystems® 3500 Series Genetic Analyzer by capillary electrophoresis.
Figure 4. Optimization of ExpiCHO FeedAddition(A) Fo r the Max Titer Protocol, additio n of s econd feed can be a dded on Day 4, 5, or 6 pos t-tr ansfectio nwith similar perfo rma nce. (B) Two equal volume fee ds on Days 1 and 5 p ost-tr ansfectio n do uble protei ntiters.
Figure 2.Workflow for IdentifyingHigh-ExpressingCHOClones(A) CHO cells were transiently tr ansfecte d and evalu ated fo r prot ein expressi on using th e ClonePixsystem (Molecula rDevices ). (B) Selec ted cl ones w ere furt her evaluat ed via tr ansfectio n with plasmidsfor multiple protei ns. Control h uman IgG titers using an existi ng CHO clone a re shown as well asControl titers for the pre-clonal pool.
B
Figure 8. Kinetics of hIgG Expression(Green line ) Standar d Protocol consisting of on e feed and n o tempe ratu re shift. (Blue line) High Tite rProtocol co nsisting of one fee d a nd temp eratu re shift to 32°C. (R ed lin e) hIgG kinetics using the MaxTiter Protocol consisting of two feeds and temperature shift to 32°C.
A
Media
Cells
Optimized Transient Expression
V. Workflow
IV. ExpiFectamineCHO Enhancer
VII. Expression Levels of Various Transient Systems
VIII. Glycan Analysis
Days in Culture
VCD (x 106cells/mL)
(solid line) %
Viability
(dotted line)
0 1 2 3 4 5 6 7 80
5
10
15
20
25
0
50
100
ExpiCHO Expression Media Attributes• One media for growth and transfection
• Formulated specifically for transient transfection• Chemically-define d (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/impo rt/e xp ort limitations
ExpiCHO-S Cell Line Attributes• Derived from GMP CHO-S cells
• Adapted for high-density culture• Non-clumpy, single-cell phenotype• Short doubling time (~17 hours)
• Stable growth and expression over 20+ passages
Figure 10. Titers in FreeStyleCHO,Expi293 andExpiCHOExpression levels of human I gG, Rabbit IgG a nd Eryth ropoi etin in FreeStyleCHO, Expi2 93 an d ExpiCHOtransient expr ession systems ar e shown . ExpiCHO titers ran ge fr om2 5x – 1 60x th ose of Fr eeStyleCHOand 2x – 4x those obtained using the Expi293 system.
hIgG Titer (mg/L)
One Feed Two Feeds
TIter (m
g/L)
Day 3
Day 5
Day 7
B
A
C
A B
A B
hIgG (m
g/L)
ExpiFectamine Enhancer
0
1000
2000
3000
4000
+ -
Figure 9. Scalability of the ExpiCHO System
160x 3x 95x 4x 25x 2x
0.14 0.28 0.43 0.57 0.71 0.860
1
2
3
Titer (g/L)
DNA Concentration (µg/mL)
Expi293 ExpiCHO Stable CHO-S0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
G0
A1
G2F
Man5A1F
G1F_2
G1F_1
G0F
D4 D5 D6 D70
1
2
Titer (g/L)
Timing of Second Feed
125mL 250mL 500mL 1L 2L 3L0
1000
2000
3000
4000
Titer (hIgG) m
g/L
125rpm 70rpm
25mL 50mL 100mL 200mL 400mL 750mL
Flask Size
Transfection Volume
Shake Speed
32°CEnhancer2 feeds
32°CEnhancer1 feed
37°CEnhancer1 feed
Days of Culture
Titer (g/L)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 140
1
2
3
Max Titer Protocol
High Titer ProtocolStandard Protocol
Expi293
ExpiCHO
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