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

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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)

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A B

A B

hIgG  (m

g/L)

ExpiFectamine  Enhancer

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+ -­

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

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3

Titer  (g/L)

DNA  Concentration  (µg/mL)

Expi293 ExpiCHO Stable  CHO-­S0%

10%

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G0

A1

G2F

Man5A1F

G1F_2

G1F_1

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D4 D5 D6 D70

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Titer  (g/L)

Timing  of  Second  Feed

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

1000

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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

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Max  Titer  Protocol

High  Titer  ProtocolStandard  Protocol

Expi293

ExpiCHO